FROM/THE 
LIBRARY 

OP 

LEONARD  WILLIAM 
*  BUCK* 


UNIVERSITY  QF  CALIFORNIA 

MEDICAL  CENTER  LIBRARY 

SAN  FRANCISCO 


LEONARD  W.  BUCK,  M.D. 


rag  '•• 


GENERAL  CHEMISTRY 


OF    THE 


ENZYMES 


BY 


HANS  EULER 

Professor  of  Chemistry  in  tlie   University  of  Stockholm 


TRANSLATED  FROM 

THE  REVISED  AND  ENLARGED  GERMAN  EDITION 
BY 

THOMAS   H.   POPE 


FIRST    EDITION 

FIRST   THOUSAND 


NEW  YORK 

JOHN   WILEY  &   SONS 

LONDON:  CHAPMAN  &  HALL,  LIMITED 
1912 


152651 


Copyright,  1912 

BY 

THOMAS   H.    POPE 


SCIENTIFIC    PRESS 

ROBERT    DRUMMOND   AND    COMPANY 
BROOKLYN,    N.   Y. 


PREFACE  TO   THE   GERMAN   EDITION 


As  the  title  of  this  book  indicates,  the  author  has  attempted 
to  review  the  more  important  facts  of  enzymology  from  a  general 
standpoint  and  to  fit  them,  so  far  as  is  possible,  into  their 
proper  places  in  the  fabric  of  general  and  physical  chemistry. 
The  aim  has  not  been  to  give  a  complete  synopsis  of  our  know- 
ledge of  the  enzymes,  for  already  several  such  summa'ries  are 
available. 

It  may  perhaps  be  asked:  Is  the  time  yet  ripe  for  giving 
a  representation  of  the  physical  chemistry  of  the  enzymes? 
The  author  feels  that  this  question  must  be  answered  in  the 
affirmative,  although  it  is  evident  that  extensive  regions  and 
important  problems  in  the  subject  are  still  entirely  untouched. 
The  period  during  which  the  marshalling  of  facts  was  the  most 
essential  task  was  followed  by  one  in  which  it  was  sought  to 
harmonize  the  somewhat  crude  and  imperfect  experimental 
data  with  the  laws  of  theoretical  chemistry.  The  deviations 
from  theory  seemed  to  be  wide  and  the  peculiarities  of  enzymic 
reactions  numerous.  Only  in  the  most  recent  times  has  the 
need  for  experimental  revision  of  the  quantitative  data  made 
itself  felt.  Improvements  have  been  effected  in  the  practical 
methods,  while  the  factors  participating  in  the  reactions  have 
become  more  clearly  understood  and  are  hence  more  fully  taken 
into  account.  It  is  now  being  found  that  the  results  obtained 
from  these  more  exact  and  comprehensive  investigations  corre- 
spond more  closely  with  those  required  to  satisfy  physico-chemical 
theories.  At  the  stage  which  has  thus  been  reached  in  the 
development  of  enzymology  a  review  such  as  that  now  pub- 
lished does  seem  to  be  justified.  The  author  has  therefore 
decided  to  allow  the  two  reports  on  this  subject  which  appeared 
in  the  "  Ergebnisse  der  Physiologic  "  in  1907  and  1910,  to  be 

iii 


iv  PREFACE  TO  THE  GERMAN  EDITION 

arranged  and  issued  in  book-form,  despite  the  fact  that  many 
problems  still  call  for  fuller  treatment. 

Although  this  monograph  is  intended  more  especially  as  an 
aid  to  scientific  research  in  enzymology,  yet  the  author  trusts 
that  it  will  be  found  useful  by  those  concerned  with  the  prac- 
tical applications  of  enzymic  actions.  Thus  an  understanding 
of  the  dynamics  of  enzyme  reactions  is  indispensable  for  the 
rational  estimation  of  enzymic  activities,  such  as  that  of  pepsin 
in  the  gastric  juice  or  that  of  diastases  in  malt,  and  these  examples 
serve  to  show  how  theory  may  be  of  value  to  the  physician  and 
to  the  technical  worker. 

An  appendix  to  the  book  contains  a  short  sketch  of  experi- 
mental methods,  more  especially  of  those  for  which  the  original 
literature  is  not  readily  accessible.  Professor  Bertrand  has 
kindly  permitted  the  insertion  of  the  tables  prepared  by  him 
for  use  with  his  admirable  method  of  estimating  reducing  sugars. 

A  considerable  part  of  the  labour  involved  in  preparing  this 
monograph  has  been  undertaken  by  Miss  Beth  af  Ugglas,  Assistant 
in  the  Biochemical  section  of  the  Chemical  Laboratory  here  and 
to  her  I  wish  to  express  my  sincere  thanks. 

H.  EULER. 
STOCKHOLM,  January,  1910. 


PREFACE  TO  THE  ENGLISH  EDITION 


ALTHOUGH  only  two  years  have  elapsed  since  the  first 
publication  of  this  work  in  the  German  language,  the  great 
energy  with  which  the  study  of  enzyme  chemistry  is  being  prose- 
cuted has  rendered  necessary  numerous  additions  and  alterations. 
In  view  of  the  results  of  recent  investigations,  some  of  the 
sections,  e.g.,  those  concerned  with  the  glucosides  and  the 
fermentation  enzymes,  have  indeed  been  entirely  rewritten. 

To  Mr.  Pope's  request  to  allow  of  the  issue  of  an  English 
edition  of  the  book  the  author  acceded  the  more  readily  because 
of  the  great  success  which  has  for  a  long  time  past  attended 
enzymological  research  in  English-speaking  countries.  At  the 
present  time,  when  various  different  paths  have  become  clearly 
marked  in  general  enzymic  chemistry,  the  opportunity  is  wel- 
comed of  laying  the  author's  views  before  English  workers  in 
this  field. 

To  Mr.  Pope  the  author  is  indebted,  not  only  for  a  careful 
translation  of  his  book,  but  also  for  certain  improvements  and 
additions  in  the  part  dealing  with  practical  methods  and  for 
the  references  to  the  literature. 

In  the  initial  treatment  of  so  extensive  a  subject  as  enzyme 
chemistry  omissions  are  scarcely  avoidable,  and  the  author 
would  be  grateful  to  any  readers  who  may  contribute,  either 
by  sending  him  copies  of  their  papers  or  by  any  other  means, 
to  render  a  subsequent  edition  more  complete. 

H.  EULER. 
STOCKHOLM,  March,  1912. 

v 


JOURNALS  REFERRED  TO  BRIEFLY 

Biochem.  Z.:  Biochemische  Zeitschrift  (Berlin). 
Chem.  Ber. :  Berichte  der  deutschen  chemischen  Gesellschaft. 
C.  R. :  Comptes  rendus  de  rAcademie  des  Sciences  (Paris). 
H. :  Hoppe-Seyler's  Zeitschrift  fur  physiologische  Chemie. 
Hofm.  Beitr.:  Hofmeister's  Beitrage  zur  chemischen  Physiologic  und 
Pathologic. 

Lieb.  Ann.:  Justus  Liebig's  Annalen  der  Chemie. 

Pfliig.  Arch. :  Pfliiger's  Archiv  fiir  die  gesammte  Physiologic. 

Soc.  Biol.:  Comptes  rendus  de  la  Socie*te*  Biologique  (Paris). 

vi      • 


CONTENTS 


PAOE 

Introduction - 1 

CHAPTER  I 

Special  Chemistry  of  the  Enzymes 5 

Nomenclature 5 

Classification 6 

Sphere  of  Action  of  the  Enzymes;    Their  Preparation  and  Puri- 
fication    7 

Esterases 9 

Enzymes  of  the  Higher  Carbohydrates 13 

'Enzymes  of  the  Glucosides  and  Disaccharides 18 

Other  Enzymes  which  Hydrolyse  Glucosides 27 

Phytase 31 

Hexosephosphatase 32 

Pectinase 32 

Qarbamases  (Proteinases) '. 33 

Pepsin 33 

Trypsin 36 

Erepsin 38 

Proteolytic  Enzymes  of  Plants 38 

Nucleases 41 

Urease 43 

Amidases  (Desamidases) 43 

Coagulating  Enzymes 45 

Chymosin 45 

Thrombin,  Fibrin-ferment 49 

Enzymes  of  Fermentation 50 

Enzymes  of  Alcoholic  Fermentation 51 

Lactic  Acid  Bacteria-zymase 58 

Oxydases 58 

Alcoholase 60 

Aldehydases 61 

Laccase 62 

Tyrosinase 64 

vi  i 


viii  CONTENTS 


Peroxydases 65 

Catalases 67 

Reducing  Enzymes  (Reductases;  Reducase) 68 

Appendix 69 


CHAPTER  II 

Physical  Properties  of  the  Enzymes 71 

Adsorption 75 

Solid,  Neutral,  Adsorption-media 81 


CHAPTER  III 

Activators  (Co-enzymes),  Paralysors  and  Poisons 90 

Kinases  of  Unknown  Composition 91 

Special  Organic  Activators 92 

Acids,  Bases,  and  Neutral  Salts 94 

Protective  Agents 115 

Inhibiting  Agents  (Paralysors) 115 

Inorganic  Salts 116 

Organic  Poisons  and  Inhibiting  Agents 118 


CHAPTER  IV 

Chemical  Dynamics  of  Enzyme  Reactions 124 

Theoretical  Principles  of  Enzymic  Dynamics 125 

Catalysis 127 

Reversible  Reactions 137 

Experimental  Data  on  the  Course  of  Enzyme  Reactions 146 

Esterases  and  Lipases 146 

Amylase 155 

Invertase 158 

Maltase 166 

Lactase 168 

Enzymes  of  Emulsin 171 

Proteolytic  Enzymes 175 

Rennet  (Chymosin) 200 

Fibrin-ferment 205 

Zymase 206 

Catalases 215 

Oxydases 219 

Peroxydases 223 

Tyrosinase 22S 

Oxidation  of  Xanthine .  .  .230 


CONTENTS  ix 


CHAPTER  V 

PAGE 

Influence  of  Temperature  and  Radiation  on  Enzymic  Reactions .   231 

Influence  of  Radiation .   245 


CHAPTER  VI 

Chemical  Statics  in  Enzyme  Reactions 251 

Equilibria 252 

End-states  and  Stationary  States 255 


CHAPTER  VII 
Enzymic  Syntheses 261 

CHAPTER  VIII 

Specificity  of  Enzyme  Action 274 

Conclusion 283 

APPENDIX 

Practical  Methods 286 

Tables  for  the  Estimation  of  Sugars  by  Bertrand's  Method 306 

Index  of  Authors 313 

Index  of  Subjects 321 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


INTRODUCTION 

THE  name  enzymes  or  unorganised  ferments  is  given  to 
animal  or  vegetable  substances  of  unknown  composition  and 
constitution  which,  in  the  organism  itself  or  even  independently 
of  the  organ  or  cells  in  which  they  arise,  are  able  to  accelerate 
chemical  reactions.  The  term  enzyme  is  thus  included  in  the 
much  more  general  term,  catalyst. 

By  catalyst  we  understand  a  substance  which,  without  being 
required  by  the  accelerated  reaction  or  appearing  among  the 
final  products,  alters  the  velocity  with  which  a  chemical  system 
strives  to  attain  its  final  condition.  But  enzymes  are — at  least 
with  the  degree  of  purity  in  which  they  have  as  yet  been  obtained 
— rarely  ideal  catalysts.  Only  with  difficulty,  however,  can  a 
limit  be  set  between  these  substances  and  ideal  catalysts,  this 
being  greatly  dependent  on  the  experimental  conditions. 

The  literature  of  the  last  few  years  shows,  indeed,  that  a 
certain  limitation  in  the  meaning  of  the  term  enzyme  is  desirable ; 
and,  as  a  rule,  those  substances  which  are  required  in  stoichio- 
metric  proportions  by  the  reactions  in  which  they  participate 
are  not  regarded  as  enzymes. 

With  non-enzymic  catalyses  the  quantity  of  the  accelerating 
substance  is  mostly  small  compared  with  that  of  the  substance 
acted  on — indeed,  an  ideal  catalyst  should  accelerate  the  trans- 
formation of  unlimited  amounts  of  "  substrate."  Even  in  the 
most  minute  quantities  some  enzymes  certainly  exert  very  con- 
siderable amounts  of  action;  but  usually  their  activity  becomes 
limited  with  lapse  of  time  and  does  not  produce  more  than  a 
certain  amount  of  change.  As  we  shall  see  later,  this  limitation  is 


2  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

due,  partly  to  the  participation  of  the  enzyme  in  the  equilibrium 
of  the  reaction  and  partly  to  the  chemical  instability  of  these 
substances.  One  property  which  the  enzymes  exhibit  and  which 
is  generally  regarded  as  characteristic  of  them,  is  that  of  becom- 
ing inactive  if  their  solutions  are  heated  for  a  longer  or  shorter 
time  at  a  high  temperature — about  100°.  This  is  not  an  absolute 
criterion,  as  inorganic  catalysts  and  enzymes  do  not  exhibit  any 
fundamental  difference  in  this  respect.  As  soon  as  any  con- 
stituent of  an  organ  which  accelerates  a  reaction  is  explained 
chemically  or  identified  with  a  known  compound,  no  reason  remains 
for  terming  it  an  "  enzyme";  whether  the  term  enzyme  is  to  be 
retained  for  such  substances,  or  whether  the  name — scientifically 
more  accurate — of  catalyst  is  to  be  employed,  is  entirely  a  matter 
for  the  future.  But  the  choice  of  a  definition  is  of  subordinate 
importance,  as  we  are,  in  many  cases,  so  far  removed  from  any 
chemical  explanation  of  the  enzymes  that  this  term  will  certainly 
persist  for  a  long  time. 

The  distinction  between  the  enzymes  and  the  toxines  is  also, 
to  some  extent,  arbitrary.  Common  to  both  classes  of  bodies 
are  their  origin  in  the  living  organism,  their  capacity  of  forming 
anti-bodies,  and  certain  other  properties,  as  also  are  their  modes 
of  action.  On  the  other  hand,  the  toxines  are  characterised  with 
moderate  sharpness  by  their  poisonous  action.  We  can,  indeed, 
omit  a  treatment  of  this  extensive  subject  all  the  more  readily, 
as  the  physical  chemistry  of  the  toxines  has  undergone  considerable 
development  during  recent  years. 

Of  far  greater  importance  than  deciding  how  the  enzymes  are 
to  be  limited  is  the  definition  of  the  physical  and  chemical  prop- 
erties of  typical  representatives  of  these  remarkable  substances. 

The  aim  in  view  is,  of  course,  the  exact  description  of  the 
enzyme  by  a  chemical  formula  and  by  constants  characteristic 
of  the  pure  substance.  For  the  general  chemistry  of  the  enzymes, 
clear  views  concerning  the  degree  of  purity  and  the  composition 
of  the  various  members  are  of  the  greatest  importance.  In  the 
first  place,  the  enzymes  must  be  prepared  and  analysed,  and  here 
physico-chemical  investigations  also  afford  valuable  aid. 

The  first  question  to  be  decided  is :  Do  the  enzymes  occur  in  a 
state  of  true  solution,  or  must  they  be  classed  with  colloidal 
substances?  Or,  speaking  more  strictly,  which  enzymes  approach 
the  one  and  which  the  other  limiting  case,  and  what  can  be 


INTKODUCTION  3 

affirmed  concerning  their  molecular  magnitude  and 
degree  of  dispersion?  As  criteria  on  these  points  serve 
diffusion,  adsorption  phenomena  and  also  behaviour  in  the  electric 
field. 

More  recent  measurements  have  shown  that  the  influence  of 
temperature  on  enzyme  action  can  be  defined  more  exactly 
than  earlier  data  would  have  led  one  to  suppose,  and  the  "  tem- 
perature of  destruction  "  and  "  optimum  temperature,"  which 
give  little  information,  are  now  replaced  by  well-defined  physico- 
chemical  magnitudes. 

Undoubted  and  considerable  success  has  followed  the  study, 
during  the  past  few  years,  of  activators  or  co-enzymes;  men- 
tion need  only  be  made  here  of  the  work  of  Harden  and  Young 
and  of  Buchner  and  Meisenheimer,  which  has  led  to 
the  discovery  of  essential  factors  influencing  alcoholic  fermenta- 
tion. Also,  with  reference  to  inactivators  remarkable 
regularities  have  been  observed,  those  concerning  the  influence  of 
configuration  calling  for  special  mention.  Further,  the  action  of 
poisons  is  now  so  far  understood  that,  in  enzyme  investigations, 
we  can  make  use  of  substances  which  prevent  bacterial  infection 
and  yet  have  no  harmful  effect  on  the  enzyme,  thus  avoiding  the 
errors  which  have  been  so  often  caused  in  work  of  this  kind  by 
insufficient  disinfection. 

Although  the  majority  of  the  anti-bodies,  so  important 
physiologically,  are  classed  among  the  toxines,  yet  such  a  large 
number  of  observations  have  been  made  on  the  anti-fer- 
ments that  these  must  not  remain  unnoticed. 

When  we  have,  in  the  first  part  of  this  book,  obtained  infor- 
mation concerning  the  chemical  facts  of  enzymology,  we 
must  turn  to  the  second  part  of  the  question:  In  what  manner 
is  a  reaction  induced  or  accelerated  by  an  enzyme  and  how  do 
enzymic  reactions  proceed? 

In  the  first  place,  we  will  consider  the  laws  of  chemical 
dynamics  which  come  into  play  in  enzymic  reactions  and,  in 
particular,  the  results  which  have  been  obtained  from  a  study  of 
catalysts.  Comparison  of  non-enzymic  reactions  with  enzymic 
changes  will  show  us  that  the  same  processes  are  being  dealt  with 
in  the  two  cases  and  that  the  deviations  from  the  classical  examples 
of  chemical  dynamics,  exhibited  by  many  enzyme  reactions,  are 
readily  explained  by  the  simple  assumption  that  enzyme  and 


4  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

substrate  unite  to  form  more  or  less  stable  complexes,  which  are 
to  be  regarded  as  the  "  active  "  molecules  and  hence  bring  about 
the  reaction.  This  assumption  is  adhered  to  all  the  more  strongly, 
because  it  corresponds  with  our  general  conception  of  the  role 
of  catalysts. 

In  the  fourteen  years  which  have  passed  since  the  synthesis 
of  isomaltose  by  maltase  was  discovered  (Croft  Hill),  the 
number  of  enzymic  syntheses  has  become  quite  considerable. 
Not  only  do  we  now  know  enzyme  reactions  which  proceed  in 
both  directions,  but  in  several  cases  the  synthetic  action  of  the 
enzyme  has  been  separated,  and  caused  to  take  place  apart,  from 
the  decomposing  action.  A  knowledge  of  these  syntheses  is 
naturally  of  the  utmost  importance  for  the  biochemistry  of 
animals  and  plants. 

For  chemistry  in  general  these  processes  are  the  more  import- 
ant, since,  as  is  well  known,  the  enzymes  are  extremely  sensitive 
towards  the  steric  configuration  of  the  substrate  and  lead  to  the 
formation  of  asymmetric  products.  The  enzymes  hence  place 
us  in  a  position  to  effect  asymmetric  syntheses. 


CHAPTER  I 

SPECIAL   CHEMISTRY   OF   THE   ENZYMES 

NOMENCLATURE 

THE  already  large  and  rapidly  increasing  number  of  enzyme 
actions  necessitates  a  rational  system  of  nomenclature.  According 
to  a  proposal  made  by  D  u  c  1  a  u  x  (see  Bourquelot,  Les 
ferments  solubles,  Paris,  1896),  the  name  of  the  enzyme  is 
derived  from  that  of  the  substance  on  which  it  acts:  for  example, 
lactase  is  the  enzyme  which  decomposes  lactose.  Unfortunately, 
many  departures  have  been  made  from  this  principle.  In  cases 
where  the  name  derived  from  that  of  the  substrate  is  not  suf- 
ficiently definite,  E.  O.  von  Lippmann  (Chem.  Ber.,  1903, 
36,  331 ;  see  also  B  u  c  h  n  e  r  and  Meisenheimer,  Chem. 
Ber.,  1905,  38,  621)  proposes  that  both  the  name  of  the  substance 
acted  upon  and  that  of  the  (principal)  product  formed  from  it 
should  be  indicated  in  the  name  of  the  enzyme;  for  example, 
amylo-maltase  would  be  the  enzyme  which  forms  maltose  from 
starch.  But  as  this  enzyme  is  not  a  maltase,  but  belongs  rather 
to  the  class  of  amylases,  it  would  be  more  convenient  to  term  it 
malto-amylase.  Names  in  general  use,  such  as  pepsin,  zymase 
and  erepsin,  are  retained.  Although  the  employment  of  a  rational 
method  of  naming  enzymes  is  to  be  desired,  yet,  on  the  one  hand, 
the  right  to  give  or  alter  a  name  must  be  left  with  the  discoverer, 
and,  on  the  other,  the  region  of  action  and  the  individuality  of 
enzymes,  like  pepsin  are  not  yet  so  completely  determined  as  to 
allow  of  the  adoption  of  a  perfectly  suitable  name. 

According  to  a  suggestion  by  the  author  (H.,  1911,  74,  13), 
the  names  of  synthesising  enzymes  should  be  made  to  indicate 
the  substances  which  they  form  and  to  terminate  with  the  syllable 
"ese";  thus  phosphatese  would  be  the  enzyme  which  syn- 
thesises  organic  esters  of  phosphoric  acid,  and  nitrilese  ( B-nitrilase 
according  to  Rosenthaler)  that  which  forms  nitriles. 

5 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


CLASSIFICATION 

Since  very  little  is  known  concerning  the  nature  of  the  enzymes, 
the  classification  of  these  bodies  is  based  on  the  chemical  reactions 
which  they  induce.  And  it  is  to  be  expected  that  the  classifica- 
tion indicated  by  the  chemical  actions  would  also  be  brought  out 
in  the  physical  and  chemical  properties  of  the  enzymes. 

An  enumeration  of  all  the  enzymes  described  in  the  literature 
of  the  subject  does  not  fall  within  the  scope  of  this  work;  indeed, 
there  are  a  very  large  number  of  such  substances,  the  individuality 
of  which  has  not  been  sufficiently  well  established. 

The  following  summary  serves  rather  to  indicate,  in  a  general 
way,  typical  reactions  in  which  enzymes  play  a  part. 


Reac- 
tion. 

Substrate. 

Products. 

Enzyme. 

r 

Esters  : 

Fatty  acids  +alcohols 

Esterases: 

Fats 

Higher  fatty  acids  +glycerol 

Lipases 

Lower  esters 
Chlorophyll  +alcohol 

Lower  fatty  acids  +alcohols 
Crystalline  chlorophyll  +phytol 

Butyrases 
Chlorophyl- 

luse 

Higher  carbohydrates  : 

Cellulose 

Cellulase 

Hemicellulose 

Cytase 

f  Amylases  and 

Starch,  glycogeu 

Maltose  (dextrins) 

j       amylo-pec- 

[      tinases 

Inulin 

Fructose 

Inulinase 

Pectoses 

Pectin 

Pectase 

Glucosides  including 

Polysaccharides  : 

Hexoses  and  glucoside-residues 

a-Glucosides 

Glucose 

a-Glucosidase 

—    1 

/3-Glucosides 

Glucose          +sugar,  alcohol  or 
phenol-residue 

(Maltase) 
0-Glucosidase 
(Emulsin) 

2 

/3-Galactosides 

Galactose 

Lactase 

T3 

>> 

Fructosides 

Fructose  +sugar  residues 

Invertase 

n 

Other  glucosides 

Other  sugars  +phenols,  etc. 

Rhamnase, 

Myrosin,  etc. 

Phytin 

Inositol  +phosphoric  acid 

Phytase 

Hexosephosphate 

Hexose  +phosphate 

Hexosephos- 

phatase 

Digallic  acid 

Gallic  acid 

Tannase 

(Tannin) 

Carbamide  derivatives: 

R-CO-NH-R' 

R-COOH+R'-NH2 

Carbamases, 

proteinases 

Proteins 

Albumoses,  peptones 

Pepsin,  pap- 

ain 

Proteins,  albumoses, 
peptones,  peptides 

>  Peptides,  amino-acids 

/  Trypsin,  erep- 

^      sin 

Arginine 

Urea  +ornithine 

Arginase 

I 

Nucleic  acids 

Nuclein  bases  +phosphoric  acid 

Nuclease 

SPECIAL  CHEMISTRY  OF  THE  ENZYMES 


Reac- 
tion. 

Substrate. 

Products. 

Enzyme. 

Acid  amides: 

1 

Urea 

Carbon  dioxide  +NHS 

Urease 

i  . 

Amines  : 

Desamidases 

•6 

Amino-acids 

Hydroxy-acids  +NHs 

Desamidase 

W 

Guanine 

Xanthine  +NH, 

Guanase 

Adenine 

Hypoxanthine  +NHj 

Adenase 

=  .  n 

Hydrogen  peroxide 

Molecular  oxygen  +H*O 

Catalases 

|!l  ' 

Hydroxvnitriles 

Aldehyde  +HCN 

Nitrilases 

,  * 

Benzaldehyde  +HCN 

Mandelic  acid  nitrite 

d-Oxynitrilese 

=  ?.    , 

Na2HPO4  +carbohy- 

Ester   of    carbohydrate  —phos- 

003 

drate 

phoric  acid 

Phosphatese 

ill? 

Peroxides 

f  Reduction  products  of  per-  \ 
\      oxides  +O 

Peroxydases 

Sgo 

>, 

ll|| 

Casein 

Paracasein  (  +whey-albumen) 

Chymosin 

Fibrinogen 

Insoluble  fibrin 

Fibrin  ferment 

•?!  §S  ' 

(thrombin) 

5=^|S 

Pectins 

Pectinates 

Pectinase 

Glucose 

Lactic  acid 

Zymase  of  lactic 

a 

acid  bacteria 

o 

Glucose,  fructose,  man- 

Alcohol  +CO« 

Zymase     (sum- 

g' 

nose,  galactose 

total    of    the 

enzymes  of  al- 

8 

coholic    f  e  r  - 

mentation) 

0 

Phenols 

Quinones 

Phenolases 

§ 

Aldehydes 

Acids 

Aldehydases 

•f 

Alcohol 

Acetic  acid 

Alcoholoxy  d  ase 

3 

of  acetic  acid 

bacteria 

Certain  enzymes  which  exert  actions  other  than  those  given 
above  are  mentioned  hi  the  Appendix  to  Chapter  I. 


SPHERE   OF  ACTION   OF   THE  ENZYMES.     THEIR  PREPARATION 
AND   PURIFICATION 

In  this  section  are  given  such  data  as  appear  necessary  for 
understanding  the  general  behaviour  of  the  enzymes. 

The  dynamics  of  the  enzymes  suffers  in  considerable  measure 
from  the  disadvantage  that  we  know  nothing  of  the  composition 
of  these  bodies  and  hence  can  form  beforehand  no  idea  of  the 
chemical  processes  taking  place  during  their  action.  It  is,  there- 


8  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

fore,  all  the  more  necessary  to  investigate  experimentally  all 
the  factors  influencing  enzymic  reactions,  in  order  to  avoid  the 
danger  of  missing  a  secure  foundation  for  the  theoretical  treatment 
of  the  subject. 

Especially  would  the  author  point  out  that  it  is  no0t  possible 
to  pay  too  much  attention  to  the  preparation  and  purification 
of  enzymes  for  use  in  physico-chemical  measurements. 

Enzyme  preparations  are  obtained  either  by  subjecting  the 
organs  to  pressure  or  by  extracting  them  with  suitable  solvents. 
The  consistency  of  the  starting  material,  the  admixtures  which 
are  always  present,  the  age  and  especially  the  previous  history 
of  the  preparation,  influence  not  only  the  intensity,  but 
also  the  mode  of  action  of  the  enzyme  to  a  greater  extent  than 
many  investigators  have  supposed.  A  knowledge  of  the  material 
is  hence  indispensable  to  a  critical  examination  of  the  exper- 
imental results. 

Extracts  or  preparations  of  organs  often  exert  several  enzymic 
actions  at  the  same  time;  thus,  to  choose  an  example  from  recent 
literatune,  a  preparation  from  croton  seeds  has  been  found  by 
S  c  u  r  t  i  and  Parrozzani  (Gazzetta  Chim.  Ital.,  1907,  37, 
i,  476)  to  hydrolyse,  not  only  fats  and  esters  of  monobasic  acids, 
but  also  cane  sugar  and  proteins.  From  these  results,  it  should 
be  concluded,  not  that  an  enzyme  exists  possessing  a  general 
hydrolytic  capacity,  but  that  the  preparation  employed  contains 
several  enzymes. 

In  order  to  study  the  separate  components,  the  different 
actions  have  to  be  separated,  and  when  a  preparation  has  been 
obtained  which  exhibits  only  a  single  reaction,  it  is  termed 
biologically  pure.  There  still  remains,  however,  the 
possibility  that  the  various  stages  of  the  reaction  are 
ccelerated  by  different  constituents  of  the  enzyme.  It  has, 
for  example,  been  found  to  be  probable  that,  in  the  hydrolysis 
of  amygdalin,  three  enzymes  take  part,  one  of  them  effecting  the 
resolution  into  glucose  and  the  glucoside  of  mandelic  acid  nitrile, 
a  second  hydrolysing  the  latter  compound  to  mandelomtrile 
and  glucose,  while  the  third  decomposes  the  nitrile  into  benzal- 
dehyde  and  hydrocyanic  acid. 

Even  those  enzymes  which  are  biologically  the  purest  are 
still  very  far  removed  from  the  state  of  chemical  purity  and 
we  possess — and  on  this  stress  must  be  laid— no  certain  knowledge 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  9 

that  even  an  approximate  isolation  of  any  hydrolytic  enzyme 
has  yet  been  attained.  This  is  explained  by  the  instability  of  the 
enzymes,  which,  when  subjected  to  protracted  and  energetic 
purifying  processes,  become  inactive,  so  that  their  presence  can 
no  longer  be  detected;  and  also  by  the  extremely  small  con- 
centrations in  which  the  enzymes  always  seem  to  occur  in  nature, 
and  by  the  large  amounts  of  impurities — especially  of  colloidal 
substances — contained  in  the  extracts. 

Of  the  oxydases,  which  are  to  some  extent  stable  to  heat,  we 
have  chemical  knowledge  of  at  least  one  member. 

We  shall  see  later  that  the  behaviour  of  enzymes  towards  ex- 
ternal influences,  such  as  acids,  alkalies,  co-enzymes,  etc.,  is  often 
determined,  wholly  or  partially,  by  the  impurities  present. 

On  account  of  the  importance  which  processes  of  purification 
have  for  enzymology,  the  methods  employed  are  treated  some- 
what in  detail. 

As  may  be  again  mentioned,  consideration  of  the  whole  of 
the  literature  on  the  different  enzymes  does  not  come  within 
the  limits  of  this  work.  In  the  first  place,  investigations  will, 
of  course,  be  omitted  to  which  lasting  value  cannot  be  ascribed, 
and  no  attention  will  be  paid  to  those  dealing  with  purely  phys- 
iological questions  and  with  the  distribution  of  the  enzymes 
in  the  animal  and  vegetable  kingdoms,  since  these  are  not  directly 
connected  with  the  general  chemistry  of  the  enzymes. 

ESTERASES 

The  usual  action  of  these  enzymes  consists  in  the  hydrolysis 
of  esters.  The  enzymes  of  this  group  prove  to  be  more  or  less 
markedly  specific,  as  will  be  shown  more  in  detail  in  Chapter 
VIII.  It  must,  however,  be  mentioned  that  the  lipase  of  the 
stomach  decomposes,  not  only  true  fats,  but  also  the  lipoids, 
lecithin,  jecorin  and  protagon  (P.  Mayer,  Biochem. 
Z.,  1906,  1,  81;  Schumoff-Simanowski  and  Sieber, 
H.,  1906,  49,  50).  Also  pancreatic  juice,  according  to  A  b  d  e  r- 
h  a  1  d  e  n  and  others,  decomposes  lecithin  [but  a  negative  result 
was  obtained  by  Kalaboukoff  and  Terroine  (Soc.  Biol., 
1909,  66,  176)].  A  special  class  is  formed  by  the 

L  i  p  a  s  e  s  ,  which  resolve  more  especially  the  natural  fats, 
i.e.,  the  glycerol  esters  of  palmitic,  stearic  and  oleic  acids. 


10  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Animal  lipases  play  an  important  part  in  the  stomach 
(gastric  juice  and  mucous  membrane l),  pancreas  2  and  intestines  3 
of  the  higher  animals.  Also  serum  contains  lipases  (Neuberg 
and  collaborators)  and,  according  to  Pagenstecher  (Biochem. 
Z.,  1909,  18,  285),  this  is  the  case  with  all  the  organs,  especially 
the  liver  and  spleen,  of  the  ox.  These  animal  enzymes  decompose 
both  animal  and  vegetable  fats  and  oils. 

A  lipase  has  also  been  found  in  the  albumen  of  hens'  eggs. 
In  general,  it  is  relatively  difficult  to  obtain  active  extracts  from 
animal  organs  containing  lipase  and  Connstein  is  of  the 
opinion  that  it  is  best  to  employ  the  pancreatic  juice  of  the  crushed 
glands  themselves.  Aristides  Kanitz,  however,  seems 
to  have  prepared  active  glycerol-extracts  (H.,  1905,  46,  482), 
and  Lewkowitsch  and  M  a  c  1  e  o  d  have  worked  with 
aqueous  lipase  solutions  which  attack  neutral  fat  (Proc.  Roy. 
Soc.,  1903,  72,  31).  The  extraction  of  lipase-preparations  from 
the  pancreas  has  been  investigated  in  detail  by  D  i  e  t  z  and 
Pottevin  (Bull.  Soc.  Chim.,  1906,  [iii],  35,  693;  see  also  E. 
B  a  u  r  ,  Zeitschr.  f.  angew.  Chem.,  1909,  22,  97).  0  .  Rosen- 
h  e  i  m  (Journ.  of  PhysioL,  1910,  40)  has  recently  made  the 
interesting  observation  that  the  lypolytic  enzyme  can  be 
separated  from  its  activator  or  co-enzyme  by  mere  filtration 
of  the  glycerol  extract  of  pancreatic  lipase.  The  substance 
remaining  on  the  filter  is  sensitive  to  heat,  whilst  that  in  the 
nitrate  is  stable  to  heat.  A  mixture  of  these  two  exerts  enzymic 
action,  but  each  separately  is  inactive. 

There  is  a  great  amount  of  contradiction  among  the  results 
obtained  with  the  lipases ;  it  does,  however,  seem  established  that 
the  lipases  of  the  stomach  and  pancreas  do  not  exhibit  identical 
properties.  The  two  animal  lipases  appear  to  be  related  in  the 
same  manner  as  the  corresponding  proteolytic  enzymes,  pepsin 
and  trypsin;  but  they  both  differ  essentially  from  the  lipases  of 
seeds. 


*F.  Volhard,  Zeitschr.  klin.  Med.,  1901,  42,  414  and  43,  397;  W. 
Stade,  Hofm.  Beitr.,  1903,  3,  291;  A.  Zinsser,  Hofm.  Beitr.,  1906, 
7,  31;  A.  From  me,  Hofm.  Beitr.,  1905,  7,  51;  A.  Falloise,  Arch. 
Internat.  de  Physiol.,  1906,  3,  396,  and  1907,  4,  405. 

2H.  Engel,  Hofm.  Beitr.,  1905,  7,  77;  U  m  b  e  r  and  Br  u  g  s  c  h  , 
Arch.  f.  exp.  Path.,  1906,  55,  164. 

'  W.  B  o  1  d  y  r  e  f  f  ,  H.,  1907,  50,  394. 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  11 

Vegetable  lipases  are  obtained  from  oily  seeds,  especially 
of  Ricinus.  The  first  work  on  these  lipases  was  carried  out 
by  Reynolds  Green  (Proc.  Roy.  Soc.,  1890,  48,  370) 
and  S  i  g  m  u  n  d.  Further  contributions  to  the  knowledge 
of  them  are  due  to  Cojnnstein,  Hoyer  and  Wartenberg 
(Chem.  Ber.,  1902,  35,  2988),  Nicloux  (Soc.  BioL,  1904,  56, 
840  and  Proc.  Roy.  Soc.,  B.,  1906,  77,  454),  H.  E.  Armstrong 
(Proc.  Roy.  Soc.,  B.,  1905,  76,  606)  and  others.  A  very  good 
monograph  has  been  written  by  C  o  n  n  s  t  e  i  n  for  the  "  Ergeb- 
nisse  der  Physiologic"  (1904,  3).  E.  R  o  u  g  e  (Centralbl.  f. 
Bakt.,  1907,  II,  18,  403)  gives  a  resume  of  the  literature  deal- 
ing more  particularly  with  vegetable  lipases. 

Ricinus-lipase  is  only  active  in  relatively  strongly  acid  solu- 
tion, and  in  the  seeds,  it  is  activated  by  the  lactic  acid  present 
(Hoyer,  H.,  1906,  50,  414).  According  to  Braun  and 
Behrend  (Chem.  Ber.,  1903,  36,  1142,  1900),  the  seeds  of 
Abrus  precatorius,  which  are  nearly  related  to  Ricinus 
seeds,  decompose  fats  in  neutral  solution,  but  the  effect  is 
comparatively  slight.  Characteristic  of  the  Ricinus-enzyme,  as 
of  most  true  lipases,  is  its  insolubility  in  water;  it  is  hence  nec- 
essary to  bring  the  pressed  mass,  remaining  after  the  removal 
of  the  oil  from  the  seeds,  into  intimate  contact  with  the  fat- 
emulsion. 

As  to  the  other  sources  of  plant  lipases,  mention  may  be  made 
of  the  fungi,  both  higher  and  also  lower,  like  P  e  n  i  c  i  1 1  i  u  m 
(Gerard,  C.  R.,  1897,  124,  370;  Camus,  Soc.  BioL,  1897, 
49,  192),  Aspergillus  niger  (Camus,  loc.  cit.)  and 
especially  yeast  (Delbriick).  Lipases  have  also  been  detected 
in  numerous  bacteria;  they  cause  the  rancidity  of  butter  and  other 
natural  fats  (for  the  literature  see  Fuhrmann,  Bakterien- 
enzyme,  Jena,  1907). 

Butyrases.  Against  the  extended  application  of  the 
esters  of  the  lower  fatty  acids  and  the  monoglycerides  to  the  study 
of  the  lipases,  objection  has  often  been  raised.  In  particular, 
A  r  t  h  u  s  (Soc.  BioL,  1902,  53,  381)  and  also  D  o  y  o  n  and 
M  o  r  e  1  (C.  R.,  1902,  134,  1001  and  1254)  have  pointed  out  that 
H  a  n  r  i  o  t  's  experiments  with  monobutyrin  (Soc.  BioL,  1896, 
48,  925;  C.R.,  1896,  123,  753)  which  challenge  criticism  in  many 
directions,  give  no  information  as  to  the  presence  and  action  of 


12  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

the  true  lipases.  A  distinction  must  therefore  be  drawn  between 
the  lipases  and  esterases  (buty rases).  The  latter  enzymes, 
which  occur  abundantly  in  many  juices  and  organs  (blood-serum, 
liver,  kidneys)  decompose  not  only  the  monovalent  alkyl  and  the 
glyceryl  esters  of  the  lower  fatty  acids,  but  also  amyl  salicylate 
(H.  Chanoz  and  H.  Doyon,  Soc.  BioL,  1900,  52,  116,  717) 
and  similar  compounds.  Schmiedeberg's  histozyme 
is  perhaps  identical  with  these  enzymes. 

D  a  k  i  n  has  effected  asymmetric  ester-decompositions  by 
means  of  li ver-esterases ;  these  will  be  considered  in  Chapter 
VIII.  Detailed  studies  on  the  same  enzymes  are  due  to  K  as  tie, 
Loevenhart  and  E  1  v  o  v  e  ,  whose  quantitative  measure- 
ments will  be  referred  to  in  the  third  chapter.  They  give  the 
following  method  for  the 

Preparation  of  li  ver-esterases.  The  macerated  liver 
(10  grms.)  is  extracted  with  water  (100  c.c.)  and  the  extract 
filtered  through  a  linen  cloth.  Twenty  c.c.  of  this  extract  are 
diluted  with  72  c.c.  of  water  and  8  c.c.  of  a  0-OlN-solution  of 
hydrochloric  acid  added.  When  this  mixture  is  heated  to  40°, 
a  heavy  precipitate  of  protein  separates  and,  on  filtering,  a  clear 
golden-yellow  solution  is  obtained. 

The  action  of  pancreas-lipase  is  participated  in  by  a  co- 
enzyme  (R.  Magnus,  H.,  1904,  42,  149),  which  is  stable  at  a 
boiling  temperature  and  the  essential  constituents  of  which  are 
alkali  salts  of  the  bile  acids  (Magnus,  H.,  1906,  48,  376;  see 
also  Chapter  V). 

Among  the  esterases  must  also  be  classed 

Chlorophyllase.  This  very  interesting  enzyme,  which 
was  discovered  and  described  by  Willstatter  and  S  t  o  1 1 
(Lieb.  Ann.,  1911,  378,  18),  accompanies  chlorophyll  and  is 
wide-spread  in  its  occurrence.  The  reaction  which  it  produces 
is  an  alcoholysis. 

Chlorophyll  contains  three  carboxyl  groups,  one  of  which  is 
apparently  free,  and  the  others  esterified  with  a  methyl  and  a 
phytyl  (from  phytol)  group  respectively.  Only  the  latter  reacts 
with  the  alcohol,  and  then  only  under  the  influence  of  the  enzyme, 
the  phytoxyl  group  being  replaced  by  ethoxyl. 

"  In  its  action,  chlorophyllase  cannot  be  replaced  by  other 
esterases.  On  the  other  hand,  for  the  enzyme  found  in  the 
leaves,  chlorophyll  is  a  specific  substrate.  With  phaeophytin 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  13 

this  enzyme  does  not  react  so  well  and  with  an  ordinary  wax  no 
reaction  takes  place." 

ENZYMES   OF   THE   HIGHER   CARBOHYDRATES 

C  y  t  a  s  e  s  .  Whether  true  cellulose  is  decomposed  enzym- 
ically  is  still  uncertain.  More  is  known,  especially  from  the 
investigations  of  Mac  Gil  la  wry,  of  H.  T.  Brown  and 
Morris  (Journ.  Chem.  Soc.,  1890,  57,  497),  of  Reynolds 
Green  (Annals  of  Bot.,  1893,  7,  93)  and,  recently,  of  S  c  h  e  1- 
lenberg  (Flora,  1908,  98,  257),  of  the  action  of  cytase,  the 
substrates  of  which  are  the  hemicelluloses  and  their  reaction- 
products,  mannose  and  galactose;  also  pentose-polysaccharides 
—the  pentosans — are  decomposed,  but  it  is  not  yet  known 
whether  the  hydrolysis  is  complete.  Such  enzymes  occur  in  the 
intestines  of  herbivorous  animals,  in  wood-destroying  fungi 
(C  z  a  p  e  k  ,  Lotos,  1898,  46,  235;  Schorstein,  Centrabl.  f. 
Bakt.,  1902,  II,  9,  446)  and  in  bacteria.  Also  hydrocelluloses, 
which,  so  far  as  is  known,  are  nearly  allied  to  the  hemicelluloses, 
are  decomposed  by  cytases.  To  the  same  group  of  enzymes 
belong  caroubinase,  which  dissolves  the  caroubin  in  the 
carob  (C  e  r  a  t  o  n  i  a  s  i  1  i  q  u  a)  (E  f  f  r  o  n  t ,  C.  R.,  1897,  125, 
116)  and  the  enzyme  described  by  Bourquelot  and  H  e  r  i  s- 
s  e  y  (C.  R.,  1899,  129,  228,  391,  614;  1900,  130,  42,  340,  741)  as 
s  e  m  i  n  a  s  e  ,  which  occurs  in  lucerne,  T  r  i  g  o  n  e  1 1  a  and 
other  plants.  As  well  as  in  plants,  cellulases  or  cytases  are  found 
in  the  animal  body,  especially  in  the  intestines  of  graminivorous 
animals  (H.  T.  B  r  o  w  n  ,  Journ,  Chem.  Soc.,  1892,  61,  352),  in 
snails  (Biedermann  and  M  o  r  i  t  z  ,  Pfltig.  Arch.,  1898,  73, 
236)  and  in  fishes  (K  n  a  u  t  h  e) .  Experiments  by  the  author 
(Zeitschr.  f.  angew.  Chem.,  1912,  25,  46)  indicate  the  occurrence 
in  Merulius  lacrimans  (dry-rot  fungus)  of  an  enzyme 
which  decomposes  cellulose-dextrin. 

A  m  y  1  a  s  e  s  .  These  enzymes — more  accurately  termed 
malto-amylases — include  all  those  which  break  down  starch  and 
glycogen  forming  maltose.  Very  little  that  is  definite  can 
be  asserted  with  regard  to  the  individuality  of  the  amylases. 

After  the  view  had  been  expressed  by  Brown  and  Mor- 
r  i  s  and  by  Reynolds  Green  that  at  least  two  enzymes 


14  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

showing  different  biological  relations  are  to  be  distinguished 
(diastase  of  translocation  and  diastase  of  secretion),  Maquenne 
(C.  R.,  1906,  142,  124,  1059,  1387)  carried  out  a  series  of  notable 
investigations  which  explained  the  saccharification  from  a 
chemical  point  of  view.  Starch  consists,  indeed,  of  80-85% 
of  amylose  and  15-20%  of  amylopectin.  Amylase  attacks 
dissolved  amylose  and  also  " soluble  starch"  very  readily,  but  acts 
on  amylopectin  (starch-paste)  very  slowly.  Amylopectinase,  on 
the  contrary,  saccharifies  amylopectin  (starch-paste)  with  great 
ease.  The  diastases  of  the  very  varying  organs  of  plants  and 
animals  must  contain  both  of  these  diastatic  enzymes.  That  the 
saccharifying  and  liquefying  actions  of  the  "  diastases  "  are  often 
parallel  has  been  shown  more  especially  by  an  investigation 
made  by  Chrzascz  (Zeitschr.  f.  Spiritusind.,  1908,  31,  52). 
Another  recent  noteworthy  contribution  on  vegetable  diastatic 
enzymes  is  due  to  Butkewitsch  (Biochem.  Z.,  1908,  10, 
314).  Further  investigations  in  this  direction  are,  however, 
desirable,  as  well  as  a  more  detailed  study  of  the  diastatic 
decomposition  of  glycogen. 

Also  the  individuality  of  the  true  amylases,  apart  from  amylo»- 
pectin,  is  doubtful,  if  we  consider  the  far-reaching  decomposition 
necessary  in  order  to  pass  from  the  highly-condensed  starch 
through  the  dextrins  to  maltose.  After  Miss  T  e  b  b  (Journ.  of 
Physiol.,  1894,15,421),  Brown  and  Morris,  Rohmann 
(Chem.  Ber.,  1894,  27,  3251),  Hamburger  (Pflug.  Arch., 
1895,  60,  543)  and  B  e  i  j  e  r  i  n  c  k  (Centralbl.  f.  Bakt.,  1895, 
II,  1,  221)  had  detected,  in  the  mixture  of  enzymes  which 
saccharifies  starch,  an  enzyme  which  effects  the  transformation 
starch-to-maltose  and  another  which  further  breaks  down  the 
maltose  to  glucose,  W  i  j  s  m  a  n  (Rec.  Trav.  Chim.  Pays-Bas, 
1890,  9,  1),  Pottevin  and  others  assumed  that  the  reactions 
starch-to-dextrin  and  dextrin-to-maltose  are  also  effected  by 
separate  enzymes,1  and  recently  Ascoli  and  Bonfanti 
(H.,  1904,  43,  156)  speak  of  several  amylases. 

Occurrence.  Enzymes  which  attack  glycogen  and  starch  are, 
as  was  discovered  by  Claude  Bernard,  widespread  in  the  animal 

1 L  i  n  t  n  e  r '  8  statement  that  isomaltose  is  formed  during  the 
diastatic  conversion  of  starch  must,  after  subsequent  work,  more  especially 
by  Ling  and  Baker  (Journ.  Chem.  Soc.,  1895,  67,  702)  and  by 
Brown  and  Morris  (ibid.,  709),  be  regarded  as  disproved. 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  15 

kingdom.  Their  occurrence  in  blood-serum  which  was  detected  by  this 
investigator  has  been  examined  more  closely  by  Bial,  Pick,  and 
A  s  c  o  1  i  and  B  o  n  f  a  n  t  i  (H.,  1904,  43,  156).  According  to  N  a  s  s  e 
such  an  enzyme  occurs  in  muscle-plasma,  and  this  was  confirmed  by 
Halliburton  (Journ.  of  Physiol.,  1887,  8,  182).  Carlson  and 
Luckhardt  (Amer.  Journ.  of  Physiol.,  1908,  23,  148)  have  found 
amylase  in  numerous  other  body-liquids.  The  fact  that  saliva  dissolves 
starch  has  been  known  much  longer,  and  this  property  was  ascribed  by 
L  e  u  c  h  s  in  1831  to  an  enzyme,  ptyalin.  Foster  and  von 
W  i  1 1  i  c  h  also  found  amylolytic  enzymes  in  a  great  number  of  organs. 
In  addition  to  the  liver  and  pancreas,  the  muscles  are  especially  rich  in 
an  enzyme  which  attacks  glycogen;  and  Mendel  and  S  a  i  k  i  (Amer. 
Journ.  of  Physiol.,  1908,  21,  64),  by  experiments  on  the  pig,  found  that 
this  is  the  case  in  the  embryo  state,  while  other  organs  of  different 
animals  usually  become  richer  in  enzyme  as  development  proceeds 
(P  u  g  1  i  e  s  e  and  others).  According  to  Roger  (Soc.  Biol.,  1908, 
64,  1137),  an  amylase  occurs  also  in  hens'  eggs  (white  and  yolk)  and  is 
partially  soluble  in  ether. 

Of  no  less  biological  importance  than  animal  amylases  are 
those  of  plants.  The  starch-decomposing  action  of  germinated 
barley  was  discovered  by  Kirchoff  as  long  ago  as  1814. 
An  enzyme-preparation  was  made  in  1833  by  P  a  y  e  n  and 
Persoz  and  was  named  "  diastase";  this  term  is  also  much 
used  at  the  present  time,  but,  in  the  interests  of  as  rational  as 
possible  a  nomenclature,  it  should  be  replaced  by  the  terms 
amylase,  amylopectinase,  etc.1  The  whole  of  the  saccharifying 
enzyme-preparation  together,  that  is,  the  mixture  of  amylase, 
dextrinase,  etc.,  may  meanwhile  be  called  diastase.2 

In  accordance  with  the  function  of  the  diastases  of  effecting  the 
metabolism  of  the  polysaccharides,  these  enzymes  are  widespread  in 
all  parts  of  plants,  and  are  especially  abundant  in  shoots  and  leaves,  par- 
ticularly with  the  Leguminosse  and  grasses  (Brown  and  Morris, 

1  W  i  j  s  m  a  n  '  s  nomenclature  is  by  no  means  an  acceptable  one.     If 
two  enzymes  really  take  part  in  the  formation  of  maltose,  they  should  be 
distinguished  as  amylase  and  dextrinase. 

2  It  is  most  desirable  that  the  use  of  the  term  "diastases"  as  a  generic 
name  for  enzymes  should  be  abolished  from  the  French  literature.     For 
this  use  of  the  term  there  is,  indeed,  a  historical  explanation,  but  there  is 
no  justification  for  its  continuance,  especially  as  it  often  gives  rise  to  mis- 
understanding. 


16  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Journ.  Chem.  Soc.,  1893,  63,  604).  Amylases  have  further  been  detected 
in  potatoes  and  the  sugar-beet;  also  in  germinating,  starch-containing 
pollen-grains  (Reynolds  Green),  in  the  bark  of  many  plants 
(Butkewitsch,  Biochem.  Z.,  1908,  10,  314),  in  the  sap,  in  many 
higher  and  lower  fungi,  especially  in  several  species  of  yeast — here  the 
diastase  may  be  related  to  the  glycogen-content — and  finally  in  bacteria. 

Special  mention  must  be  made  of  the  so-called  t  a  k  a  -  d  i- 
a  s  t  a  s  e,  the  mixture  of  saccharifying  enzymes  from  A  s  p  e  r  - 
gillus  o  r  y  z  a  e ,  a  fungus  contained  in  koji-yeast.  It  sac- 
charifies starch  and  indeed,  according  to  Stone  and  Wright, 
and  T  a  k  a  m  i  n  e ,  more  energetically  than  does  malt-diastase. 
Experiments  with  the  view  of  preparing  it  in  a  pure  state  were 
made  byWroblewski  (Chem.  Ber.,  1898,  31,  1130). 

Preparation.  Of  the  animal  amylases,  the  ptyalin  of 
saliva  is  the  best  suited  for  preparation.  According  to  J. 
C  o  h  n  h  e  i  m  (Virch.  Arch.,  1865,  28,  241)  the  saliva  is  pre- 
cipitated with  freshly-prepared  calcium  phosphate.  From  the 
precipitate  the  ptyalin  is  dissolved  by  means  of  water,  and  the 
aqueous  solution  precipitated  with  alcohol.  Another  method 
is  given  by  K  r  a  w  k  o  w  (J.  Russ.  Phys.  Chem.  Soc.,  1887,  19, 
387),  who  precipitates  saliva-diastase  by  ammonium  sulphate. 
Direct  precipitation  of  the  saliva  with  alcohol  also  yields  a  sac- 
charifying preparation. 

C  o  h  n  h  e  i  m  has  found  saliva-diastase  to  be  free  from 
protein,  but  he  does  not  state  on  what  absolute  quantities  of 
the  preparation  the  tests  were  made. 

Von  Wittich  takes  up  pancreas-diastase  in  anhydrous 
glycerol. 

Larger  and  purer  yields  of  amylase  are  obtained  from  vege- 
table material.  From  malt  Lintner  prepared  diastase  as  fol- 
lows: one  part  of  green  malt  (or  air-dried  malt)  was  extracted 
for  24  hours  with  2-4:  parts  of  20  Jo  alcohol,  the  extract  being 
precipitated  with  2-5  times  its  volume  of  absolute  alcohol  and 
the  precipitate  washed  with  absolute  alcohol  and  ether. 

Loew  (Pflug.  Arch.,  1882,  27,  203;  1885,  36,  170)  steeps  ger- 
minated barley  in  a  little  water  and  then  extracts  with  4% 
alcohol.  He  precipitates  the  extract  with  lead  acetate,  suspends 
the  precipitate  in  water,  removes  the  lead  from  the  solution  by 
means  of  hydrogen  sulphide,  and  finally  precipitates  the  diastase 
with  a  mixture  of  alcohol  and  ether. 


SPECIAL   CHEMISTRY  OF  THE  ENZYMES  17 

0  s  b  o  r  n  e  and  Campbell  (Journ.  Amer.  Chem.  Soc., 
1896,  18,  536)  and  also  Wroblewski  (H.,  1897,  24,  73)  salt 
out  the  diastase  with  ammonium  sulphate. 

E  f  f  r  o  n  t  (Enzymes  and  their  Applications,  London  and 
New  York,  1902,  pp.  104  e  t  s  e  q.)  proposes  the  extraction  of  malt 
with  water  and,  in  order  to  diminish  the  quantity  of  the  extractive 
material  possessing  no  diastatic  action,  he  induces  alcoholic 
fermentation  in  the  infusion  by  yeast  previously  rendered  very 
poor  in  nitrogen.  E  f  f  r  o  n  t  states  that  the  fermentation 
destroys  a  large  quantity  of  carbohydrates,  removes  considera- 
ble quantities  of  proteins  and  salts,  and  leaves  the  diastase 
absolutely  untouched. 

Wroblewski  (Chem.  Ber.,  1897,  30,  2289)  gives  the 
following  method: 

Finely-ground  malt  is  extracted,  first  with  70%,  and  then' 
twice  with  45%  alcohol.  Sufficient  strong  alcohol  is  added  to 
the  last  two  extracts  to  bring  the  alcohol-content  to  70%.  The 
precipitate  formed  is  washed  with  absolute  alcohol  and  ether  and 
dried  in  a  vacuum. 

Like  O  s  b  o  r  n  e  and  Campbell  (loc.  ei  t.),  Wrob- 
lewski (Chem.  Ber.,  1898,  31,  1130)  effected  further  purifica- 
tion by  salting  out  with  ammonium  sulphate. 

Wroblewski  considered  that,  as  a  result  of  these  exper- 
iments, he  had  shown  with  certainty  that  diastase  is  a  protein 
substance  nearly  allied  to  the  albumoses,  whilst,  according 
to  T.  B.  O  s  b  o  r  n  e  (Chem.  Ber.,  1898,  31,  254),  diastase  is  a 
protein-like  substance  or  "a  compound  of  an  albumin  with  a 
proteose."  For  his  most  active  preparation  he  gives  the  fol- 
lowing composition  (calculated  for  ash-free  substance):  C,  52-5; 
H,  6-72;  S,  1  -90;  N,  16-10%.  The  solution  gives  the  character- 
istic reactions  of  the  proteins.  Wroblewski 's  purest 
preparation  had  a  nitrogen-content  of  16-5%. 

If,  however,  the  recent  researches  of  S.  Frankel  and 
Hamburg  (Hofm.  Beitr.,  1906,  8,  389)  should  be  confirmed, 
diastase  contains  neither  protein-groups  nor  reducing  sugars. 
The  non-enzymic  substances  were  precipitated  with  lead  acetate, 
the  solution  sterilised  by  filtration  and  further  purified  by  fer- 
mentation with  yeast  rendered  poor  in  nitrogen  and  subsequent 
filtration  through  a  Pukall  filter.  After  drying  in  a  vacuum,  the 
syrupy  liquid  yields  a  powder  free  from  fermentable  and  reducing 


18  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

sugars  and  from  protein.  It  represents  a  very  active  substance 
which  does  not  give  the  biuret  reaction  or  reduce  F  e  h  1  i  n  g  '  s 
solution  but  shows  a  faint  M  i  1 1  o  n  '  s  reaction;  it  also  gives 
M  o  1  i  s  c  h  '  s  reaction  and  the  pentose  reaction  slightly.  When 
dialysed  into  spring-water,  the  dissolved  diastases  are  separated 
into  two  principal  groups:  the  saccharifying  diastases  diffuse 
through  the  membrane,  whilst  the  liquefying  ones  remain. 

A  distinct  advance  seems  to  have  been  made  by  H.  C.  S  h  e  r- 
m  a  n  and  M.  D.  Schlesinger  (Journ.  Amer.  Chem.  Soc., 
1911,  33,  1195).  They  found  that  pancreas-diastase  keeps  well 
in  50%  alcohol,  and  they  purified  such  a  solution  by  dialysis. 
A  very  active  preparation  (saccharifying  power,  5000  on  Lintner's 
scale  at  40°)  contained  53-0%  C,  6-6%  H  and  15-6%  N. 

Inulinase.  In  addition  to  hemicelluloses,  starch  and 
glycogen,  another  carbohydrate,  inulin,  also  occurs  as  a  reserve 
material.  The  enzyme,  inulase  or  inulinase,  accompanying  this, 
decomposes  inulin  into  its  simplest  com- 
ponent, fructose.  Starch  is  not  attacked  by  inulinase. 

ReynoldsGreen  (Annals  of  Bot.,  1888,  1,  223)  discovered  this 
enzyme,  which  occurs  in  many  of  the  Compositse,  in  the  tubers  of  H  e  1  i  - 
anthus  tuberosus  (artichoke).  Bourquelot  (Bull.  Soc. 
Mycol.,  1893, 9,  230;  1894, 10,  49)  detected  inulinase  in  Aspergillus 
n  i  g  e  r  and  isolated  it  from  the  mycelium  of  this  fungus.  The  enzyme 
appears  to  be  widespread  in  the  Eumycetes;  Dean  (Bot.  Gaz.,  1903, 
35,  24)  found  it  also  in  Penicillium  glaucum.  The  best 
medium  for  its  action  is  0-001%  hydrochloric  acid. 

The  enzymes  which  decompose  trisaccharides,  such 
as  melicitase,  etc.,  have  not  yet  been  sufficiently  individu- 
alised. 


THE  ENZYMES  OF  THE  GLUCOSIDES  AND  DISACCHARIDES 

E.  Fischer  has  stated  that  the  disaccharides  may  be 
regarded  as  glucosides  and  can  be  classified  with  these  according 
as  they  contain  the  glucose  in  the  a-  or  (3-form.  From  the  results 
of  E.  F.  Armstrong  (Journ.  Chem.  Soc.,  1903,  83,  1305) 
and  C.  S.  H  u  d  s  o  n  (Journ.  Amer.  Chem.  Soc.,  1909,  31,  1242), 
it  appears  that  a-glucosides,  which  were  originally  characterised 


SPECIAL   CHEMISTRY  OF  THE  ENZYMES  19 

by  the  fact  that  they  are  hydrolysed  by  a  constituent  of  yeast- 
extract,  generally  yield  a-glucose. 

p-Glucosides  are  hydrolysed  by  a  component  of  almond 
extract,  and  from  such  a  glucoside  Hudson  obtained  ^-glucose. 

a-G  lucosidase;  maltase.  As  a-glucosidases  will  be 
designated  those  enzymes  which  hydrolyse  a-glucosides  specifically. 
They  are  therefore  limited,  on  the  one  hand,  by  ^-glucosidase, 
which  acts  only  on  ^-glucosides,  and,  on  the  other,  by  lactase  and 
invertase,  which  accelerate  the  hydrolysis  of  the  ^-galactosides 
or  fructosides. 

Occurrence.  In  both  the  animal  and  vegetable  kingdoms, 
maltase  almost  always  accompanies  the  diastases,  from  which  it  cannot 
often  be  separated.  Thus,  this  enzyme  has  been  found  in  blood  and  in 
serum  (G 1  e  y  and  B  o  u  r  q  u  e  1  o  t ,  Soc.  BioL,  1895,  47,  247;  Ham- 
burger, Pfliig.  Arch.,  1895,  60,  543;  Tebb,  Journ.  of  Physiol., 
1894,  15,  421 ;  Fischer  and  N  i  e  b  e  1 ,  Sitzungsber.  K.  Akad.  Berlin, 
1896,  73) ;  also  in  many  tissues  (Shore  and  Tebb,  Journ.  of  Physiol., 
1892,  13,  19),  especially  in  the  liver,  intestines  and  pancreas.  As  Miss 
Tebb  found,  the  maltase  can  be  extracted  from  these  organs — in 
both  the  fresh  and  dried  states — by  means  of  chloroform  water;  the  oppo- 
site statement  of  Brown  and  Heron  (Proc.  Roy.  Soc.,  1880,  30, 
393)  is  thus  contradicted. 

The  maltases  occur  in  great  abundance  in  the  vegetable  kingdom. 
Their  occurrence  in  m  a  1 1  and  in  y  e  a  s  t  must  -be  especially  mentioned. 
These  two  maltases  do  not  appear  to  be  absolutely  identical  (Fischer, 
H.,  1894,  26,  74).  The  lactic  acid  yeasts  and  also  kephir-grains  always 
contain  lactases  in  place  of  maltases.  Saccharomyces 
Marxianus  contains  no  maltase,  but  only  invertase  (E.  C. 
Hansen;  E.Fischer  and  P.  Lindner,  Chem.  Ber.,  1895, 
28,  984).  Excepting  in  this  case,  it  can  be  said  that  invertase  always 
accompanies  maltases  in  yeast-extracts;  according  to  Beijerinck 
and  to  E.  Fischer  and  P.  Lindner  (Chem.  Ber.,  1895,  28,  984), 
invertase  is  lacking  in  Saccharomyces  octosporus.  In 
Saccharomyces  apiculatus  neither  maltase  nor  invertase 
is  found. 

Preparation.  As  starting  material  for  the  preparation 
of  the  yeast-enzymes,  maltase  and  invertase,  it  is  best  to  employ 
pure  cultures.  The  yeast  is  used  as  fresh  as  possible  and  is 
well  pressed  and,  according  to  E.  Fischer  (H.,  1898,  26,  74), 
ground  and  well  shaken  two  or  three  times  with  the  ten-fold 
quantity  of  water.  Removal  of  the  mother-liquor  is  effected 


20  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

most  suitably  by  a  P  u  k  a  1 1  flask-filter.  The  yeast  is  pumped 
as  dry  as  possible  and  is  then  spread  out  in  as  thin  a  layer  as 
possible  on  poro.us  tiles  and  dried  in  the  air  at  the  ordinary  tem- 
perature. Under  these  conditions  it  gradually  shrivels  up  and 
assumes  a  dark-grey  colour.  After  1-2  days,  it  is  powdered  as 
finely  as  possible  and  left  to  dry  in  the  air  until  it  forms  a  loose 
powder.  The  final  drying  may  also  be  carried  out  at  30-35°. 
In  this  state  the  yeast  can  be  kept  for  months  without  the  maltase 
being  destroyed.  When  the  enzyme  is  to  be  used,  the  yeast 
is  extracted  with  10-15  times  the  quantity  of  water  for  12-20 
hours  at  30-35°,  with  occasional  shaking,  the  liquid  being  then 
filtered  through  paper.  Toluene  serves  as  a  suitable  antiseptic. 

R  6  h  m  a  n  n  (Chem.  Ber.,  1894,  27,  3251)  heated  the  yeast 
for  an  hour  at  105^110°  before  extraction,  but  this  procedure, 
according  to  Croft  Hill,  is  not  to  be  recommended.  The 
latter  investigator  gives  the  following  method  (Journ.  Chem. 
Soc.,  1898,  73,  636) :  Good,  pressed  bottom-yeast  is  washed  three 
times  with  distilled  water  by  decantation,  collected  on  a  covered 
filter,  spread  out  on  a  porous  support  and  dried  in  a  vacuum 
over  sulphuric  acid.  The  yeast  dries  in  about  two  days  and  is 
then  powdered  and  sieved  through  a  cloth,  a  yellowish-white 
powder  being  obtained.  This  powder  is  then  spread  out  on  a 
double  layer  of  fine  tulle  over  the  mouth  of  a  glass  vessel  in  an 
oven  previously  heated  to  40°.  In  successive  quarters  of  an 
hour,  the  temperature  is  raised  to  60°,  70°,  90°  and  100°,  the 
last  being  maintained  for  15  minutes,  after  which  the  prepara- 
tion is  allowed  to  cool  in  a  desiccator.  The  yeast  is  then  weighed, 
ground  in  a  mortar  with  10  times  its  weight  of  0-1%  sodium 
hydroxide  solution,  filled  into  flasks  with  addition  of  toluene 
and  left  at  the  room-temperature  for  3  days.  The  extract  is 
now  filtered,  first  through  paper  and  afterwards  through  a  Cham- 
berland  filter.  If  1  c.c.  of  this  fresh  extract  is  added  to  20  c.c. 
of  2%  maltose  solution  at  30°,  about  20%  of  the,  sugar  is 
hydrolysed  in  40  minutes. 

Different  yeasts  appear  to  contain  widely  varying  proportions 
of  maltase,  so  that  not  every  species  of  yeast  is  suitable  for  the 
preparation  of  maltase. 

Trehalase.  Trehalose,  a  disaccharide  composed  of  two 
molecules  of  glucose,  is  hydrolysed  by  an  enzyme  which  was 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  21 

found  by  Bourquelot  in  Aspergillus  and  other  fungi, 
by  Fischer  (H.,  1898,  26,  79)  in  the  diastase  of  green  malt 
and  in  yeasts  of  the  Frohberg  type,  and  by  Kalanthar  (H., 
1898,  26,  97)  in  various  other  yeasts.  Whether  the  enzyme, 
which  acts  best  with  a  very  slight  concentration  of  hydrogen-ions, 
really  differs  from  maltase  is  not  yet  established ;  Bourquelot 
(Soc.  Biol.,  1895,  47,  515)  regards  it  as  a  separate  enzyme,  but 
Fischer  is  not  in  agreement  with  this  opinion. 

The  ^-glucosidases  hydrolyse  (3-methylglucoside  and 
also  most  of  the  natural  glucosides,  which  are  on  this  account 
placed  in  the  ^-series.1 

It  has  been  shown  recently  by  Hudson  and  Paine  (Journ. 
Amer.  Chem.  Soc.,  1909,  31,  1242)  that  the  hydrolysis  of  a  typical 
natural  glucoside,  salicin,  under  the  influence  of  emulsin,  yields 
^-glucose.  It  is  better  here  not  to  apply  the  ordinary  principles 
of  nomenclature,  but  to  name  the  glucosides  according  to  the  form 
of  glucose  to  which  they  give  rise  and  the  enzymes  so  that  they 
refer  to  the  glucosides  characterised  in  this  way. 

Of  special  glucosido-glucoses  hydrolysed  by  emulsin,  mention 
may  be  made  of: 

Isomaltose  (E.  Fischer,  Chem.  Ber.,  1895,  28,  3024; 
compare  also  E.  F.  Armstrong,  Proc.  Roy.  Soc.,  B,  1905, 
76,  592). 

Gentiobiose  (Bourquelot  and  H  e  r  i  s  s  e  y,  C.R., 
1902,  135,  399). 

Cellose  or  Cellobiose  (E.  Fischer  and  G. 
Z  e  m  p  1  e  n,  Lieb.  Ann.,  1909,  365,  1). 

C  e  1 1  a  s  e  .  From  the  results  of  fractional  filtration  of 
extracts  of  Aspergillus  niger  by  Holderer's  method, 
G.  B  e  r  t  r  a  n  d  and  M.  H  o  1  d  e  r  e  r  (C.  R.,  1909,  149,  1385 
and  1910,  150,  230)  assume  the  existence  of  an  enzyme  which 
differs  from  (3-glucosidase  and  acts  specifically  on  cellose.  An 
enzyme  extracted  from  apricot  seeds  hydrolyses  only  cellose  and 
not  trehalose. 

xThe  opportunity  must  not  be  neglected  of  pointing  out  that  E. 
Fischer,  who  introduced  this  method  of  reasoning,  has  issued  a  warning 
that  it  must  not  be  regarded  as  absolutely  safe  (Lieb.  Ann.,  1909,  365,  1): 
"For  it  might  be  assumed  that  one  and  the  same  enzyme  hydrolyses  both 
the  alcohol-glucosides  and  the  glucosido-glucoses.  But  as  long  as  no  pure 
enzyme  is  obtained,  complicated  mixtures  like  emulsin  or  yeast-extract 
having  to  be  used,  no  proof  of  this  exists." 


22  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Cellase  occurs,  together  with  other  enzymes,  in  apricot  kernels, 
almonds,  barley,  and  the  mycelium  of  Aspergillus. 

^-Methylgalacto  sides  are  also  hydrolysed  by 
the  enzymes  of  the  almond  (E.  Fischer,  Chem.  Ber.,  1895, 
28,  1429)  and,  since  Fischer  found  that  this  enzyme  likewise 
effects  the  hydrolysis  of  lactose,  the  latter  is  to  be  regarded  as  a 
p-galactoside.  But  we  shall  not  go  far  wrong  if  we  assume, 
with  B  o  u  r  q  u  e  1  o  t  and  H  e  r  i  s  s  e  y  (C.  R.,  1903,  137,  56) 
and  with  E.  Fischer,  that  this  latter  action  is  not  brought 
about  by  the  same  enzyme  as  hydrolyses  (S-glucosides,  but 
depends  on  the  presence  of  an  enzyme  which  decomposes  (3- 
galactosides  and  is  hence  either  identical  with,  or  nearly  related 
to,  the  lactase  occurring  in  lactose-yeasts.  The  view  that  kephir- 
lactase  and  emulsin- lactase  are  different,  has  been  advanced  by 
H.  E.  and  E.  F.  Armstrong  and  E.  H  o  r  t  o  n  (Proc.  Roy. 
Soc.,  B,  1908,  80,  321).  They  assume  that  the  one  enzyme  is  a 
galacto-lactase  and  the  other  a  gluco-lactase,  the  first  being  taken 
up  by  the  galactose-residue  and  the  latter  by  the  glucose-residue 
of  milk-sugar. 

A  preparation  which  is  biologically  purer  is  obtained,  accord- 
ing to  Pottevin  (Ann.  Inst.  Pasteur,  1903,  17,  31),  from 
Aspergillus  niger,  Aspergillu  s-emulsin  hydrolys- 
ing  only  ^-glucosides  and  not  (3-galactosides  or  milk-sugar. 

Amygdalin  is  resolved  by  the  enzymes  of  the  almond 
into  glucose,  benzaldehyde  and  hydrocyanic  acid,  and  mandelo- 
nitrile  glucoside,  formed  by  the  action  of  yeast-enzymes  on 
amygdalin,  is  also  hydrolysed  by  emulsin  into  its  simplest  com- 
ponents (E.  Fischer,  Chem.  Ber.,  1895,  28,  1508).  The 
glucosido-glucose  contained  in  amygdalin  is  not  identical  with 
maltose,  since,  on  the  one  hand,  maltose  is  not  liberated  by  the 
action  of  emulsin  (C  a  1  d  w  e  1 1  and  Courtauld,  Journ. 
Chem.  Soc.,  1907,  91,  666;  R  o  s  e  n  t  h  a  1  e  r,  Arch,  der  Pharm., 
1908,  245,  684)  and,  on  the  other,  maltose  has  no  retarding  action 
on  the  hydrolysis  by  emulsin  (A  u  1  d ,  Journ.  Chem.  Soc.,  1908, 
93,  1276). 

It  is,  therefore,  best  to  indicate  by  "emulsin"  the  mix- 
ture of  glucoside-resolving  enzymes  and  to  characterise  the  prepara- 
tion according  to  its  origin,  a  distinction  being  drawn  between 
Aspergillus  -emulsin,  almond  -emulsin,  etc. 


SPECIAL  CHEMISTKY  OF  THE  ENZYMES  23 


For  the  enzyme-constituents,  rational  names  are  then  chosen, 
the  results  of  H.  E.  and  E.  F.  Armstrong  and  H  o  r  t  o  n  , 
C  a  1  d  w  e  1  1  and  Courtauld,  and  Rosenthaler 
indicating  at  least  four  components  of  almond-emulsin,  namely: 

(1)  Amygdalase,  characterised  by  the  reaction: 


C6H5  •  CH(CN)  -  O  -  CoHioO4  •  O  • 

Amygdalin 

=  C6H5  •  CH(CN)  •  0 

Mandelonitrile  glucoside 

(2)  A  (3-glucosidase,    which  acts  on  g-glucosides,   among 
them  mandelonitrile  glucoside: 

C6H5  •  CH(CN)  •  O  •  C6HiiO5+H2O 

Mandeloaitrile  glucoside 

=  C6H5  •  CH(CN)  -  OH+C6Hi206. 

Mandelonitrile  Glucose 

(3)  A    hydroxynitrilase: 

C6H5-CH(CN)-OH   =   C6H5-CHO   +  HCN. 

Mandelonitrile  Benzaldehyde       Hydrocyanic  acid 

In  addition  to  these  three  substances  which  take  part  in  the 
decomposition  of  amygdalin,  the  existence  in 
emulsin  must  be  assumed  of: 

(4)  An    enzyme    which    resolves    milk-sugar,    i.e.,    a   lactase 
(which   Armstrong    terms   gluco-lactase). 

According  to  N  e  u  b  e  r  g  (Ergeb.  der  Physiol.,  1904,  3,  446), 
the  conjugated  glycuronic  acids  are  also  decomposed  by  emulsin. 

The  synthetic  action  of  certain  components  of  emulsin  is 
treated  more  in  detail  in  Chapter  VII. 

Occurrence.  1.  Phanerogams.  As  well  as  in  almonds,  emul- 
sin is  found  in  the  leaves  of  Prunus  laurocerasus  (where 
laurocerasin  likewise  occurs),  in  the  seeds  of  many  of  the  Rosacese,  in 
manihot  (Guignard)  and  in  extracts  of  numerous  plants,  such 
asMonotropa,  Polygala  [Bourquelot,  Journ.  de  Pharm. 
et  Chim.,  1904,  (5),  30,  433],  Malus  communis,  Hedera 
helix,  etc.  (H  e  r  i  s  s  e  y  ,  Thesis,  "  Recherches  sur  I'Emulsine," 
Paris,  1899). 

2.  Cryptogams.  It  was  discovered  simultaneously  in  P  e  n  i  c  i  1  - 
Hum  glaucum  by  Gerard  (Soc.  Biol.,  1893,  45,  651)  and  in 
Aspergillus  niger  by  Bourquelot,  who  also  detected 
enzymes  capable  of  attacking  glucosides  in  many  other  fungi,  especially 


24  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

in  the  Polyporus  species  found  in  wood.  H  6  r  i  s  s  e  y  has  found 
emulsin  in  many  lichens  and  mosses.  Bourquelot  has  recently 
observed  hydrolysis  of  otherwise  unknown  glucosides  by  emulsin  (Arch, 
der  Pharm.,  1907,  245,  172).  Fermi  and  Montesano  (Centralbl. 
f.  Bakt.,  1894,  I,  15,  722),  Gerard  (Soc.  Biol.,  1896,  48,  44)  and 
Twort  (Proc.  Roy.  Soc.,  B,  1907,  79,  329)  have  detected  emulsin  in 
bacteria,  27  species  out  of  44  examined  having  the  property  of  hydro- 
lysing  glucosides. 

Worthy  of  note  is  the  observation  of  Henry  and  A  u  1  d  (Proc. 
Roy.  Soc.,  B,  1905,  76,  568)  that  many  yeasts  also  exhibit  "emulsin" 
action. 

Animal  enzymes  closely  related  to  emulsin  were  found  by  G  e*  r  a  r  d 
(Soc.  Biol.,  1896,  48,  44)  in  the  kidneys  of  the  horse  and  rabbit.  In 
molluscs  B  i  e  r  r  y  and  G  i  a  j  a  (Soc.  Biol.,  1906,  58,  1038)  found 
enzymes  capable  of  hydrolysing  populin  and  phloridzin;  extracts  of 
cross-spiders  also  resolve  amygdalin  (Robert  and  W.  Fischer). 

Decompositions  of  glucosides  by  animal  extracts  were  also 
noted  byGonnermann  (Pfliig.  Arch.,  1904,  103,  225;  1906, 
113,  168)  and,  more  recently  by  K  o  b  e  r  t ;  according  to  the 
latter,  extract  of  placenta  hydrolyses  amygdalin,  arbutin,  salicin 
and  helicin. 

Preparation:  Herissey  (Thesis;  compare  Bour- 
quelot, Arch,  der  Pharm.,  1907,  245,  172). 

One  hundred  grams  of  sweet  almonds  are  steeped  for  about 
a  minute  in  boiling  water  and,  after  draining,  are  carefully  peeled. 
They  are  then  ground  as  finely  as  possible  in  a  mortar  without 
water,  the  product  obtained  being  macerated  at  room-temperature 
with  200  c.c.  of  a  mixture  of  equal  parts  of  distilled  water  and 
water  saturated  with  chloroform.  After  about  24  hours,  the 
mass  is  strained  and  pressed  through  a  damp  cloth.  This  pro- 
cedure yields  150-160  c.c.  of  liquid,  to  which  10  drops  of  glacial 
acetic  acid  are  added  to  precipitate  the  casein.  The  clear  nitrate 
(120-130  c.c.)  is  added  to  500  c.c.  of  95%  alcohol,  the  precipitate 
thus  formed  being  collected  on  a  smooth  filter  and,  after  draining, 
treated  with  a  mixture  of  equal  volumes  of  alcohol  and  ether. 
After  drying  in  a  vacuum  over  sulphuric  acid,  horny,  transparent 
plates  are  obtained  and,  when  ground,  these  give  an  almost 
white  powder. 

Invertase  (  =  a-Fructosidase) .  Invertin  or  sucrase  owes 
its  name  to  its  property  of  converting  cane-sugar  into  invert- 


SPECIAL  CHEMISTRY  OF  THE   ENZYMES  25 

sugar  (  =  glucose + fructose).  The  sphere  of  action  of  invertase 
extends  to  all  synthetic  a-methylfructosides;  on  the  other  hand,, 
^-methylfructoside,  a-glucosides  and  a-galactosides  resist  its 
action.  Apart  from  these  synthetic  glucosides,  gentianose — a 
trisaccharide  from  Gentiana  lute  a — is  also  attacked  by 
invertase,  which  resolves  it  into  fructose  and  g  e  n  t  i  o  - 
b  i  o  s  e.  Further,  melitriose  (raffinose),  a  trisaccharide  occurring 
in  the  sugar-beet,  is  decomposed  by  invertase,  yielding  fruc- 
tose and  m  e  1  i  b  i  o  s  e  . 

Occurrence.  The  distribution  of  invertase  in  the  yeasts  is  as 
well  known  as  important;  in  the  majority  of  cases,  it  is  accompanied  by 
maltase  and,  in  the  lactose-yeasts,  by  lactase.  Invertase  occurs  alone 
in  only  few  yeasts,  among  them  being  Saccharomyces  Marxi- 
anus  (Fischer,  H.,  1898,  26,  75).  S.  apiculatus  contains  no 
invertase. 

Of  other  lower  organisms  which  contain  invertase,  mention  may  be 
made  of  Fusarium,  Streptococcus  (Leuconostoc) 
mesenterioides,  Aspergillus  oryzae  and  M  o  n  i  1  i  a 
Candida.  A  long  series  of  invertase-containing  bacteria  is  also 
known,  especially  owing  to  the  investigations  of  Fermi  and  M  o  n  - 
t  e  s  a  n  o  (Centralbl.  f.  Bakt.,  1895,  1,  482,  542). 

With  the  higher  plants,  invertase  is  found  especially  in  the  green 
leaves  and  young  shoots  (K  a  s  1 1  e  and  Clark,  Amer.  Chem.  Journ., 
1903,  30,  422),  in  ripe  bananas,  in  mulberries,  in  resting  and,  still  more 
abundantly,  germinating  pollen,  and  in  wheat  and  barley  embryos;  in 
the  crown  leaves  of  Robinia  viscosa  and  pseudacacia, 
Papaver  rhoeas,  Rosa  species  and  Bougainvillea 
bracts.  Also  in  fruits,  such  as  dates,  which,  when  unripe,  contain  the 
invertase  as  an  insoluble  endo-enzyme,  this  only  becoming  soluble  when 
the  fruit  ripens  (V  i  n  s  o  n  ,  Journ.  Amer.  Chem.  Soc.,  1908,  30,  1005). 

Invertase  is  found  in  human  intestinal  juice,  even  immediately  after 
birth  (Kriiger),  but  not  in  that  of  cattle  (F  i  s  c  h  e  r  and  Niebel). 
Robertson  (Edinburgh  Med.  J.,  1894)  found  it  in  almost  all  organs. 

Preparation.  If  yeast-cells  are  to  be  extracted  with 
water,  it  is  first  of  all  necessary  to  kill  them,  either  by  treatment 
for  a  short  time  with  ether  or  for  a  longer  time  with  alcohol 
(Osborne,  H.,  1899,  28,  399),  etc.,  or  by  heating  the  dry  yeast 
at  105°  (S  a  1  k  o  w  s  k  i)  or  by  plasmolysis  (I  s  s  a  e  w). 

After  careful  dehydration  in  a  vacuum  and  subsequent  heat- 
ing, yeast  may  yield  about  12%  of  its  invertase  on  extraction 
(Euler  and  Kullberg,  H.,  1911,  73,  94). 


26  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Living  yeast  also  gives  up  invertase  to  the  surrounding  liquid 
— water  or  sugar  solution — but  in  relatively  small  quantities. 
Presumably  it  is  more  especially  the  old  cells  from  which  the 
invertase  can  be  extracted  directly. 

0  '  S  u  1 1  i  v  a  n  and  T  o  m  p  s  o  n  left  top-fermentation  beer-yeast 
for  a  month  at  15°  so  that  it  became  completely  liquid;  [it  was  then 
pressed  and  the  clear  solution  obtained  precipitated  with  47%  alcohol. 
The  precipitate,  after  deposition,  was  dissolved  in  water  and  sufficient 
alcohol  added  to  bring  its  content  in  the  liquid  up  to  28%;  in  this 
solution  the  invertase  remained  dissolved,  whilst  the  majority  of  the 
protein  substances  separated.  On  raising  the  alcoholic  content  of  the 
filtered  liquid  to  47%,  a  precipitate  was  again  formed  and  this  was 
washed  with  absolute  alcohol  and  dried  in  a  vacuum.  The  preparation 
thus  obtained  was  very  active,  but  still  not  quite  pure;  it  contained 
about  5%  of  ash  (magnesium  and  potassium  phosphates),  which  the 
English  investigators  regarded  as  an  admixture.  Their  further  puri- 
fication experiments  showed,  as  had  already  been  indicated  by  the  work 
of  Osborne  (H.,  1899,  28,  399)  and  of  Salkowski,  that  invertase 
is  not  a  protein.  Even  the  purest  preparation  contains,  besides  phos- 
phoric acid,  a  carbohydrate.  Wroblewski  regards  this  as  an 
impurity,  as  also  does  0  shim  a  (H.,  1902,  36,  42);  the  latter  came 
to  the  conclusion  that  yeast-gum  consists  of  a  substance  which  contains 
d-mannose  and  a  methyl-pentosan  giving  fucose  on  hydrolysis. 

H  a  f  n  e  r  ,  who  carried  out  a  thorough  examination  of  pure  invertin 
(H.,  1904,  42,  1),  regards  it  as  by  no  means  disproved  that  this  peculiar 
carbohydrate  always  adhering  to  invertin  is  an  integral  constituent  of 
the  enzyme.  A  large  part  of  the  phosphorus  of  invertin  preparations  is 
combined  organically.  The  specific  activity  of  the  enzyme  is  not  con- 
nected with  the  presence  of  large  nitrogenous  groups  like  the  albumoses 
or  peptones;  the  absence  of  peptones  is  also  supported  by  the  failure 
of  the  biuret  action.  The  nitrogen  is  probably  present  in  the  form  of 
smaller  groups,  which  have,  however,  not  been  investigated. 

Very  active  invertase  solutions  are  also  obtainable  from 
pure  cultures  ofAspergillus  niger. 

The  best  method  for  preparing  invertase  in  as  pure  a  form 
as  possible  consists  in  removing  protein  by  lead  acetate  and 
kaolin,  and  in  subsequently  applying  the  following  diffusion 
process  (E  u  1  e  r  and  Kullberg,  H.,  1911,  73,  335): 
Bottom  fermentation  beer-yeast  is  subjected  to  autolysis  for 
3-10  days  and  then  precipitated,  as  Hudson  recommended, 
with  excess  of  lead  acetate;  the  whole  mass  is  then  ground  with 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  27 

kaolin  and  the  liquid  pumped  off.  The  lead  is  precipitated  by 
means  of  hydrogen  sulphide  and  the  nitrate  ground  several 
times  with  kaolin  and  a  little  charcoal  and  filtered.  By  means  of 
a  collodion  dialysor,  the  enzyme  solution  is  freed  from  the  impuri- 
ties, which  diffuse  rapidly,  and  is  finally  precipitated  with  alcohol. 

Two  kilos  of  pressed  yeast,  treated  in  this  way,  give  about 
8  grms.  of  a  pure  white  powder,  which  is  freed  from  further 
quantities  of  nitrogenous  impurities  by  dialysis  or  diffusion. 
The  preparations  are  protein-free,  and  the  content  of 
nitrogen  varies  between  0-3  and  2%.  The  molecular 
weight  exceeds  25,000. 

The  activity  is  d=0°  =  10  minutes,  i.e.,  0-05  grm.  of 
the  preparation,  dissolved  in  25  c.c.  of  an  8%  cane-sugar  solu- 
tion,  reduces  the  rotation  of  the  cane-sugar  to  zero  in  10  minutes 
at  a  temperature  of  20°. 

The  sensitiveness  of  invertase  to  temperature  (cf.  Chapter 
V)  is  such  that  the  activity  of  an  invertase  solution  is  diminished 
by  one-half  by  heating  for  30  minutes  at  63°  (H  .  E  u  1  e  r  and 
af  Ugglas).  Its  optimum  temperature  is  53-56°.  The 
influence  of  the  acidity  of  the  solution  on  the  velocity  of  inver- 
sion has  been  investigated  in  detail  by  Sorensen  and  by 
Hudson  (cf.  Chapter  IV). 

A  poisonous  action  towards  invertase  is  shown  by  mercury 
salts  and  potassium  cyanide  (and  nearly  all  salts  with  an 
alkaline  reaction);  hydrocyanic  acid  and  chloroform  are  less 
harmful,  whilst  thymol  and  toluene  are  without  effect. 


OTHER  ENZYMES  WHICH  HYDROLYSE   GLUCOSIDES 

The  number  of  different  individuals  in  this  group  seems  to 
be  very  large,  but  the  sphere  of  action  and  specificity  of  the 
enzymes  described  are  usually  very  indefinite.  Closely  related 
to  g-  glucosidase  is: 

Gaultherase  or  betulase,  the  specific  action  of 
which  consists  in  hydrolysing  the  glucoside  of  methyl  salicylate 
(gaultherin) .  Neither  salicin  nor  amygdalin  is  attacked  by  this 
enzyme. 

Occurrence.  Exclusively  in  plants.  It  was  discovered  by 
Schneegans  (Arch,  der  Pharm.,  1894,  232,  437)  in  the  bark  of 


28  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Betula  lenta.  At  the  same  time  Bourquelot  found  it  in 
several  Polygala  and  Azalea  species,  and  in  Spiraea 
ulmaria,  Monotropa  hypopitys  and  Gaultheria 
procumbens  (C.  R.,  1896,  123,  315;  J.  de  Phann.  et  Chim., 
1896,  3,  577). 

Preparation,  according  to  Bourquelot  (loc.  cit.)  : 
Monotropa  plants  are  ground  with  sand  and  the  glucoside 
removed  by  digesting  for  half  an  hour  with  95%  alcohol.  The 
residue,  which  contains  the  enzyme,  is  quickly  dried  with  alcohol 
and  ether,  after  which  the  enzyme  can  be  extracted  with  water. 
Cf.  B  e  i  j  e  r  i  n  c  k  (Centrabl.  f.  Bakt.,  1899,  II,  5,  325). 

Schiitzenberger  mentioned  an  enzyme  which  hydro- 
lyses  populin  and  also  phillyrin,  a  glucoside  occurring  in  the 
bark  of  Phillyrea  latifolia,  but  he  did  not  investi- 
gate it  further. 

Sigmund  (Monatsh.  f.  Chemie,  1909,  30,  77)  found  an 
enzyme,  which  decomposes  salicin  but  does  not  seem  to  be 
identical  with  emulsin,  in  certain  species  of  S  a  1  i  x  and 
P  o  p  u  1  u  s  ;  he  also  found  one  which  hydrolyses  arbutin  in 
Calluna  vulgaris  and  Vaccinium  myrtillus. 
W  .  Sigmund  (Monatsh.  f.  Chemie,  1910,  31,  657)  dis- 
covered an  enzyme,  capable  of  hydrolysing  sesculin,  in  the  seed- 
coats  of  the  horse-chestnut  (Aesculus  hippocastanum). 
It  does  not  appear  to  be  either  an  amygdalase  or  a  lipase,  but  is 
not  yet  sufficiently  defined.  Sigmund  proposes  for  it  the  names, 
salicase,  arbutase  and  aesculase. 

According  to  T.  Weevers  (Rec.  Trav.  bot.  J^Teerland., 
1910,  8)  an  enzyme  which  hydrolyses  salicin  specifically  occurs 
in  Salix  purpurea  and  Populus  monilifera,  and 
one  that  hydrolyses  arbutin  in  Vaccinium  vitis  idaea 
and  Pinus  communis. 

B  i  e  r  r  y  and  G  i  a  j  a  (Soc.  BioL,  1907,  62,  1117)  found, 
in  snails  and  Crustacea,  an  enzyme  which  is  not  identical  with 
"  emulsin  "  but  which  hydrolyses  populin  and  phloridzin.  It 
remains  to  be  shown  that  these  enzymes  are  not  really  general 
g-glucosidases. 

G  e  a  s  e  is  the  name  given  by  Bourquelot  and  H  e  r  i  s  - 
s  e  y  (C.  R.,  1905,  140,  870)  to  a  specific  enzyme  from  G  e  u  m 
urbanum  (Herb  Bennett)  and  r  i  v  a  1  e  which 
liberates  eugenol  from  a  glucoside  contained  in  these  plants. 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  29 

Elaterase,  from  Ecballium  elaterium  hydro- 
lyses  elaterin  (Berg). 

Rhamnase  hydrolyses  xanthorhamnin,  yielding,  according 
to  G  .  and  C  h  .  Tanret  (Bull.  Soc.  Chim.,  1899,  [iii],  21, 
1065),  rhamninose  and  rhamnetin.  Rhamninose  is  regarded  as 
a  trisaccharide,  which  can  be  hydrolysed  into  2  mols.  of  rhamnose 
(methylpentose)  and  1  mol.  of  galactose.  The  enzyme  occurs  in 
Rhamnus  infectoria. 

Besides  these  glucoside-enzymes,  another  series  is  known 
which  hydrolyse  glucosides  of  one  or  the  other  group  in  a  specific 
manner. 

M  y  r  o  s  i  n  decomposes  sinigrin  or  potassium  myronate  into 
glucose,  potassium  hydrogen  sulphate  and  allyl  mustard  oil 
(allyl  isothiocyanate)  according  to  the  equation: 

CioH18OioNS2K  =  C3H5-CNS  +  C6Hi206  +  KHS04 

Sinigrin  Allyl  mustard  oil  Glucose 

Also  other  sulphur-glucosides  occurring  in  the  Cruciferse  are 
hydrolysed  by  myrosin;  but,  according  to  E.  Fischer 
(Chem.  Ber.,  1894,  27,  3483),  a-  and  ^-glucosides  are  not 
attacked. 

Occurrence.  The  distribution  of  myrosin  has  been  shown  by 
the  investigations  of  Spatzier  (Pringsheim's  Jahrb.  f.  wiss.  Bot., 
1893,  25,  39)  and,  especially,  of  G  u  i  g  n  a  r  d  (C.  R.,  1890,  111,  249 
and  920;  also  Journ.  de  Bot.,  1894,  67  and  85).  It  is  characteristic  of 
the  Cruciferse  and  certain  allied  families  and  is  found  also  inManihot- 
species.  It  is  localised  in  certain  cells  which  are  rich  in  proteins  and 
are  dispersed  through  the  tissues.  G  u  i  g  n  a  r  d  has  isolated  mechan- 
ically such  cells  and  cell-layers,  e.g.,  the  pericycle  of  Cheiranthus. 
Roots  contain  the  enzyme  mainly  in  the  cork,  whilst,  in  the  stem,  it  is 
met  with  especially  in  the  pericycle.  Leaves  are  often  very  rich  in 
myrosin,  which  occurs  in  the  young  mesophyll. 

Poisons  and  antiseptics  :  The  action  of  myrosin  is 
prevented  by  tannin  in  a  concentration  of  1%  or  by  salicylic  acid  in 
solutions  stronger  than  1-5%.  Chloral  in  1%  concentration  is  less 
harmful,  and  borax  quite  harmless.  Cf.  Reynolds  Green,  "  Soluble 
Ferments  and  Fermentation,"  1899,  p.  154. 

Erythrozyme  is  the  name  given  to  an  enzyme 
(S  c  h  u  n  c  k  ,  1852)  which"  decomposes  the  ruberythrin  or 
ruberythric  acid  of  madder  into  alizarin,  dihydroxyanthra- 


30  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

quinone  and  glucose.  This  hydrolysis  is  also  effected,  although 
more  slowly,  by  emulsin,  with  a  constituent  of  which,  ery- 
throzyme  is  closely  allied  or  identical. 

Indigo-enzymes.  Breandat  founa  in  the  leaves 
of  Isatis  alpina,  an  enzyme  which  decomposes  i  n  d  i  - 
can,  the  glucoside  of  indoxyl,  into  indoxyl  and  a  sugar  (indi- 
glucin).  And  according  to  Beijerinck  (Malys  Jahrb., 
1900)  an  enzyme  exists  capable  of  hydrolysing  the  allied  isatan 
(from  Isatis  tinctoria). 

L  o  t  a  s  e  from  Lotus  arabicus  decomposes  the 
glucoside  lotusin  into  lotoflavin  (1  :  3  :  3'  :  5' — tetrahydroxy- 
flavone),  glucose  and  hydrocyanic  acid.  According  to  D  u  n  - 
stan  and  Henry  (Proc.  Roy.  Soc.,  1900,  67,  224,  and  1901, 
68,  374),  it  is  different  from  emulsin. 

Phaseolunatase,  investigated  by  Dunstan, 
Henry  and  Auld  (Proc.  Roy.  Soc.,  5,  1907,  79,  315)  seems 
to  be  identical  with  the  linamarase  of  Jorissen  and 
Hairs  (Bull.  Acad.  Roy.  Belgique,  1891,  21,  518)  and  possibly 
with  maltase. 

As  mentioned  on  p.  6,  lactase  decomposes  milk-sugar 
into  d-galactose  and  d-giucose  and,  according  to  E.  Fischer, 
it  hydrolyses  p-galactosides  generally.  This  action  appears  to 
be  strictly  specific,  since  neither  ex-  nor  (3-glucosides  are  attacked. 
Maltase  and  lactase  are  found  in  various  yeasts  (E  .  Fischer, 
H.,  1898,  26,  81)  and  fungi  (B  o  u  r  q  u  e  1  o  t  and  H  e  r  i  s  - 
sey),  and  scarcely  ever  occur  together.  Eurotiopsis 
G  a  y  o  n  i  forms  an  exception  to  this  rule,  as,  according  to 
Laborde  (Ann.  Inst.  Pasteur,  1897,  11,  1),  it  attacks  both 
maltose  and  lactose. 

Occurrence.  Its  existence  in  lactose-yeasts  was 
assumed  by  Beijerinck  (Centralbl.  f.  Bakt.,  1889,  6,  44)  but  was 
first  definitely  proved  by  E  .  F  i  s  c  h  e  r  (Chem.  Ber.,  1894,  27,  3481). 
In  the  animal  organism  it  does  not  occur  very  largely.  It  is  found  in 
both  the  freshly  macerated  placenta  and  in  the  dry  powder.  Human 
intestinal  mucus,  as  well  as  that  of  the  calf  and  dog,  contain  lactase, 
but  only  with  the  young  organism.  In  the  intestinal  juice  of  adult 
man  no  lactase  is  found  (Hamburger  and  H  e  k  m  a,  J.  de  Physiol. 
et  Pathol.  g£n.,  1902,  4,  805).  According  to  Plimmer  (Journ.  of 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  31 

PhysioL,  1906,  35,  20)  lactase  always  occurs  in  carnivora  and  omnivora, 
but  with  herbivora,  with  the  exception  of  the  rabbit,  it  is  found  only 
in  the  young  animal.  W  e  i  n  1  a  n  d  (Zeitschr.  f .  Biol.,  1899,  38,  606) 
found  this  enzyme  in  the  pancreas.  Lactase  must  also  occur  in  all  those 
bacteria  which  decompose  milk-sugar  with  formation  of  lactic  acid  and 
alcohol,  the  hexoses,  which  are  formed  as  intermediate  products,  result- 
ing from  the  action  of  this  enzyme ;  Fuhrmann  (Vorlesungen  liber 
Bakterienenzyme,  p.  96)  mentions  especially  Bacillus  acidi 
laevolactici  and  Bacterium  coli. 

Preparation.  (Fischer,  Chem.  Ber.,  1894,  27, 
2991  and  3481) .  Air-dried  yeast  is  carefully  ground  with  powdered 
glass,  the  mass  being  then  digested  with  20  times  its  quantity 
of  water  for  20  hours  at  30-35°  and  filtered  through  a  P  u  k  a  1 1 
filter.  The  enzyme  solution  prepared  in  this  way  undoubtedly 
possessed  the  property  of  converting  milk-sugar  into  hexoses, 
but  its  action  was  weaker  than  that  of  the  aqueous  extract  of 
kephir-grains. 

M  e  1  i  b  i  a  s  e  .  Melibiose,  a  product  of  the  hydrolysis  of 
ramnose,  can  be  further  hydrolysed  to  d-galactose  and  d-glucose, 
so  that  it  contains  the  same  components  as  lactose.  This 
hydrolysis  may  be  effected  by  an  enzyme  occurring  in  certain 
bottom-yeasts,  but  not  in  top-fermentation  yeasts.  The 
enzyme  is  closely  allied  to  maltase  and  is  perhaps  to  be  regarded 
as  a  maltase  (Fischer,  H.,  1898,  26,  81). 

PHYTASE 

An  enzyme  which  decomposes  phytin  or  inositolhexaphos- 
phoric  acid,  C6Ho[OPO(OH)2]6,  into  inositol  and  phosphoric 
acid,  was  obtained  by  N.  Suzuki,  Yoshimura  and 
Takaishi  (Bull  Coll.  Agric.,  Tokyo,  1907,  7,  503)  from 
rice-  and  wheat-bran.  The  preparation  itself  was  free  from 
phosphorus  and  hydrolysed  neither  amylose  nor  proteins.  Pre- 
sumably the  enzyme,  like  phytin,  is  widespread  in  the  vegetable 
kingdom.  According  to  M  c  C  o  1 1  u  m  and  Hart  (Journ. 
of  Biol.  Chem.,  1908,  4,  497),  phytase  is  also  contained  in  the 
liver  and  blood,  but  not  in  muscle-  or  kidney-extract.  Quite 
recently,  A.  W.  D  o  x  and  Ross  Golden  (Journ.  of  Biol. 
Chem.,  1911,  10,  183)  have  detected  phytase  in  lower  fungi. 


32  GENERAL  CHEMISTRY  OF  THE  ENZYMES 


HEXOSEPHOSPHATASE 

According  to  Harden  and  Young  (Proc.  Roy.  Soc., 
B,  1910,  82,  327),  this  enzyme,  which  occurs  in  pressed  yeast 
juice  and  also  in  yeast  dried  at  room-temperature,  separates  the 
phosphoric  acid  from  hexosephosphate.  See  later  under 
"  zymase." 

PECTASE 

It  is  undoubtedly  most  rational  to  employ  the  name  p  e  c  - 
t  a  s  e  s  for  those  enzymes  which  convert  pectoses  into  pectin 
and  pectinic  acids.  The  reaction,  which  yields  also  arabinose, 
consists  certainly  of  a  hydrolysis,  but  the  details  of  the  chemical 
changes  occurring  are  unknown.  The  pectinic  acids  formed 
exist  in  the  plants  as  calcium  salts.  The  work  of  M  a  n  g  i  n 
(C.  R.,  1888-1893)  and  of  Devaux  (Soc.  phys.  nat.  de 
Bordeaux,  3)  can  only  be  mentioned  here.  The  enzyme  here 
termed  pectase  was  obtained  from  malt-extract  by  B  o  u  r  - 
quelot  and  Herissey  (C.  R.,  1898,  127,  191;  1899, 
128,  1241),  who  called  it  pectinase;  according  to  the 
general  principle  of  naming  the  enzyme  after  the  substrate, 
this  should  be  altered  to  pectase. 

PECTINASE 

By  the  term  pectinase  should  be  indicated  the  enzyme  which 
coagulates  dissolved  pectin-substances,  e.g.,  in  fruit-juices,  in 
presence  of  lime,  to  gelatinous  calcium  salts  of  the  feebly  acid 
pectinic  acids.  Here  also  there  is  as  yet  no  explanation  of  the 
chemical  reaction  taking  place.  A  high  concentration  of  acid 
retards  or  completely  prevents  coagulation,  which,  in  the  case 
of  acid  fruit-juices,  proceeds  only  after  neutralization  with  lime. 
The  velocity  of  the  reaction  is  conditioned  by  a  certain  equilib- 
rium between  the  enzyme  and  the  concentrations  of  the  acid 
and  calcium  salts.  Without  the  action  of  the  enzyme,  the 
lime  is  unable  to  coagulate  pectins  to  calcium  pectates; 
soluble  calcium  salts  can,  indeed,  induce  pectins  to  coagulate, 
but,  in  this  case  another  product  is  formed,  namely  a  pectinate 
soluble  in  0-2%  hydrochloric  acid.  Calcium  pectate,  however, 
yields  insoluble  pectinic  acid  with  0-2%  hydrochloric  acid.  It 


SPECIAL  CHEMISTKY  OF  THE  ENZYMES  33 

should  be  noted  that  Bertrand  and  M  a  1 1  e  v  r  e  ,  to 
whom  is  due  a  thorough  investigation  of  this  enzyme  (C.  R., 
1894,  119,  1012;  1895,  120,  110,  and  121,  726),  named  the  latter 
pectase. 

Occurrence.  It  is  found  in  nearly  all  plants,  especially  in 
young,  quickly-growing  organs,  shoots,  leaves,  roots,  and  fruit.  In 
great  abundance  and  in  an  extremely  active  condition,  the  enzyme 
appears  in  the  extract  of  clover  or  lucerne,  and  also  in  the  leaves  of  the 
potato  plant,  rape,  etc. 

CARBAMASES  (P r ot einases) 

Three  proteolytic  enzymes  or  groups  of  enzymes  are  dis- 
tinguished: the  pepsin  of  the  gastric  secretion,  the  trypsin  of 
the  pancreas  and  the  erepsin  of  the  intestinal  mucus.  It  is 
very  probable  that  these  three  substances  are  not  individuals, 
but  rather  mixtures  of  enzymes,  which  yet  act  specifically  on 
certain  protein  complexes.  These  three  enzyme-groups  can, 
however,  be  readily  differentiated  according  to  their  origin. 
More  difficult  is  the  division  of  the  vegetable  proteinases,  where 
classification  is  not  possible  according  to  either  the  localisation 
of  the  enzymes  or  the  media  in  which  they  act.  Hence,  a  dis- 
tinction between  vegetable  pepsins  and  vegetable  trypsins  can 
hardly  be  drawn.  The  only  classification  at  present  apparent 
depends,  on  the  one  hand,  on  a  separation  of  the  peptases  from 
the  true  proteinases,  and,  on  the  other,  on  a  limitation  of  the 
pepsinases,  for  which  the  absence  of  the  lower  hydrolytic  products 
is  characteristic.  It  is,  however,  doubtful  whether  trypsin 
itself  carries  the  hydrolysis  further  than  pepsin  does,  or  whether 
it  owes  this  property  to  an  accompanying  peptase.  In  any 
case,  a  strict  division  of  the  proteinases  is  at  present  difficult. 

Pepsin:  Pepsinases 

The  decomposition  of  proteins  effected  by  pepsin  extends, 
so  far  as  is  known,  to  all  proteins;  the  products  formed  con- 
sist of  albumoses  and  peptones,  so  that  the  hydrolysis 
is  incomplete,  lower  polypeptides  and  amino- 
acids  not,  as  a  rule,  appearing  (Abder- 
h  a  1  d  e  n  and  R  o  s  t  o  c  k  i  ,  H.,  1905,  44,  265). 


34  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Occurrence:  In  the  gastric  juice  of  all  the  vertebrates  examined, 
with  the  exception  of  certain  fishes.  The  pepsin  of  Brunner's  glands  is 
closely  allied  to  gastric  pepsin.  In  the  gastric  mucous  membrane, 
pepsin  occurs  not  in  an  active  form  but  as  a  pro-enzyme  (pepsinogen). 
It  exists  with  new-born  children  and,  with  certain  herbivorous  animals, 
even  in  the  foetal  state,  in  the  mucous  membrane,  but  is  not  present 
at  birth  in  those  carnivora  which  have  been  examined,  namely,  the  dog 
and  the  cat.  Proteolytic  enzymes,  similar  to  pepsin  and  active  in 
acid  solution,  have  also  been  found  in  several  invertebrates,  but,  with 
some  animals  at  least,  these  enzymes  are  not  identical  with  ordinary 
pepsin. 

Preparation  :  Very  active  and  stable  pepsin-solutions 
are  obtained  by  extracting  the  gastric  mucous  membrane  with 
glycerol;  the  pepsin,  together  with  protein,  may  be  precip- 
itated from  the  extract  by  means  of  alcohol.  A  considerable 
amount  of  pepsin  can  also  be  extracted  from  this  membrane 
by  acidified  water.  The  best  starting  material  is,  however, 
pure  gastric  juice,  which  can  be  prepared  by  P  a  w  1  o  w  '  s 
well-known  method  from  gastric  fistulae. 

Attempts  at  the  purification  of  pepsin  have,  up  to  the  present, 
led  to  no  final  result,  but  Pawlow's  gastric  juice  is  the 
best  material  to  work  on  for  this  purpose.  Such  gastric  juice, 
on  freezing,  yields  a  solid  product,  which  Mme.  S  c  h  u  m  o  ff- 
Simanowski  has  called  "  granular  pepsin "  (Korniges 
Pepsin) . 

The  preparation  of  the  enzyme  in  a  pure  state  has  been 
worked  at  mainly  by  N  e  n  c  k  i  and  S  i  e  b  e  r  (H.,  1901, 
32,  231;  33,  291) -and  byPekelharing  (H.,  1902,  35,  8). 

According  to  Pekelharing's  method,  P  a  w  1  o  w  '  s 
gastric  juice  is  dialysed  for  20  hours  at  0°,  the  pepsin  thus  being 
deposited  in  transparent  granules-  at  the  bottom  of  the  dialyser. 
The  turbid  liquid  is  centrifuged  and  the  colourless  residue  pressed 
and  dried;  in  Pekelharing's  opinion,  this  represents 
pure  pepsin. 

The  percentage  composition  of  the  enzyme  is  found  to  be : 

C  H  N  S  Cl  P  Fe  Ash 

Nencki  and 

Sieber:  51-26     6-74     14-33     1-5      0-48     small  variable  0-57 

Pekelharing:      51-99    7-07     14-44     1-63    0-49       0  +        0-1 

According  to  Nencki  and  Sieber,  pepsin  is  com- 
bined with  lecithin.  When  boiled  with  acids,  pepsin  yields  an 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  35 

albumose  and  a  nucleo-proteid,  which  then  yields  purine-bases 
(Pekelharing  found  xanthine)  and  pentoses  (  F  r  i  e  - 
denthal,  Engelmann's  Arch.  f.  PhysioL,  1900,  24,  181). 

They  regarded  the  pepsin  molecule  as  performing  (by  three 
different  groups)  the  three  functions  of  the  gastric  juice:  protein 
digestion,  clotting  and  plastein  formation,  a  view  with  which 
Pekelharing  agreed. 

The  precipitate  formed  when  gastric  juice  is  coagulated  by 
heat  contains,  according  to  Pekelharing,  an  acid  —  pep- 
sinic  acid  —  which  has  the  percentage  composition:  C,  50-79; 
H,  7-0;  N,  14-44  and  S,l-08  and  gives  M  i  1 1  o  n  '  s  and  the 
biuret  reactions. 

Formaldehyde  does  not  act  on  "  pepsin,"  and  this  fact 
caused  Bliss  and  N  o  v  y  to  doubt  the  protein  character 
of  pepsin.  Further,  as  was  shown  by  N  e  n  c  k  i  and  S  i  e  b  e  r 
and  also  by  Pekelharing,  it  is  possible  to  prepare  pepsin 
solutions  which  vigorously  digest  proteins  but  do  not  show  the 
protein  reactions;  indeed,  this  was  shown  to  be  the  case  as 
early  as  1885  by  Sundberg  (H.,  1885,  9,  319;  cf .  B  r  u  c  k  e  , 
Wiener  Sitz.-Ber.,  1861,  43,  601). 

The  preparation  of  a  protein-free  pepsin  solution,  which 
has  an  energetic  digestive  action  but  does  not  produce  clotting, 
was  described  by  Schrumpf  (Hofm.  Beitr.,  1905,  6,  396). 
The  mucous  membrane  was  separated  from  a  fresh  pig's  stomach 
and  was  then  ground  with  kieselguhr  and  pressed  under  a  high 
pressure.  The  pressed  juice  was  clarified  by  means  of  a  Kitasato 
filter-candle  and  was  found  to  remain  clear  on  addition  of  uranyl 
acetate,  ammonium  sulphate,  etc. 

H  e  r  1  i  t  z  k  a  (Atti  Real.  Accad.  LinceL,  1904,  [v],  13,  ii, 
51)  has  deduced  a  proof  for  the  opposite  view — that  pepsin 
is  a  true  protein — from  the  fact  that,  in  absence  of  hydrochloric 
acid,  pepsin  gradually  loses  its  activity,  peptones  appearing  at 
the  same  time.  These  may,  however,  result  from  admixtures, 
so  that  the  investigation  proves  nothing  concerning  the  chemical 
nature  of  pepsin. 

As  regards  the  individuality  of  pepsin,  it  has  already  been 
indicated  that  the  same  molecule,  by  means  of  other  side-chains, 
causes  rennet-action  and  plastein-formation,  and  this  hypothesis 
is  not  in  discord  with  the  facts  at  present  known  (cf .  J  a  c  o  b  y  , 
Biochem.  Z.,  1906,  1,  53).  On  the  other  hand,  justifiable 


36  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

objections  have  been  raised  against  P  a  w  1  o  w  '  s  assumption 
that  the  rennet-action  is  a  reversal  of  the  pepsin-action  (Bang, 
H.,  1904,  43,  358;  Schmidt-Nielsen,  H.,  1906,  48, 
92;  especially  Hammarsten,  H.,  1908,  56,  18). 

From  its  chemical  characters,  pepsin  appears  to  be  an  acid, 
and  this  view  is  sustained  by  a  number  of  different  observations 
by  various  investigators.  J  a  c  o  b  y  (Biochem.  Z.,  1907,  4, 
471)  found  that  pepsin,  and  also  rennin,  are  soluble  in  alkali, 
and  adsorption  experiments  described  by  M  i  c  h  a  e  1  i  s  indi- 
cate marked  adsorption  by  basic  media.  This  view  is  also 
supported  by  the  observation  that  pepsin  migrates  to  the 
anode. 

Pepsin  is  inactive  in  an  alkaline  medium,  but  on  acidification 
it  recovers  its  activity  more  or  less  completely  according  to  the 
nature  and  duration  of  the  previous  alkalinity  (  T  i  c  h  o  m  i  - 
row,  H.,  1908,  55,  107). 

Pepsin  exerts  its  optimal  activity  at  40°  and  its  stability  is 
greater  in  acid  than  in  neutral  solutions  and  is  further  increased 
by  the  presence  of  salts. 

Trypsin:    Tryptases 

The  enzyme  of  the  pancreas,  which  is  active  in  alkaline  or 
neutral  solution,  resolves  the  proteins  into  simple  polypeptides, 
these  being  to  some  extent  further  broken  down.  It  does  not 
decompose  all  dipeptides,  but  presumably  only  those  combina- 
tions occurring  in  the  organism  (cf.  Chapter  VIII).  On  the 
other  hand,  it  can  be  stated  that  only  those  polypeptides  are 
attacked  which  contain  naturally-occurring,  optically  active 
amino-acids.  Acid-amides,  hippuric  acid,  etc.,  are  not  attacked 
(Gulewitsch,  H.,  1899,  27,  540;  Sc.hwarzschild, 
Hofm.  Beitr.,  1903,  4,  155;  Fischer  and  Bergell, 
Chem.  Ber.,  1903,  36,  2592  and  1904,  37,  3103).  With  nucleo- 
proteins  the  protein  component  is  separated  from  the  nucleic 
acid  and  decomposed  further.  From  certain  nucleo-proteins, 
the  phosphoric  acid  is  liberated  (  B  a  y  1  i  s  s  ,  Arch.  Sci.  Biol. 
St.  Petersburg,  1904,  11,  Suppl.,  281). 

Not  at  all  improbable  is  the  assumption  of    Schaeffer 

%  and    T  e  r  r  o  i  n  e    (J.  de  Physiol.  et  Pathol.  gen.,   1910)  that 

the  trypsin  of  the  pancreas  is  accompanied  by  an  erepsin  which 


SPECIAL  CHEMISTRY   OF  THE  ENZYMES  37 

attacks   directly  .(without   kinase)    all    substances   split   off   by 
the  gastric  juice. 

Occurrence.  Trypsin  occurs  in  the  pancreatic  juice  and  in 
the  tissues  of  the  pancreatic  glands  as  a  pro-enzyme,  which  is  trans- 
formed by  specific  activators  or  kinases  into  the  active  condition. 

Trypsins  or  tryptases  have  been  found  in  all  the  vertebrata 
tested  for  them.  H  e  d  i  n  (Journ.  of  Physiol.,  1903,  30,  155  and  195) 
discovered  a  tryptic  enzyme  in  normal  blood-serum ;  it  acts .  in  an 
alkaline  medium  and  decomposes  casein,  gelatine,  and  coagulated  serum- 
albumen,  but  globulins  and  coagulated  egg-albumen  are  not  attacked. 
Besides  in  the  pancreas,  animal  tryptases  are  found  in  the  urine,  and  in 
the  spleen  and  other  organs;  also  in  hens'  eggs. 

Enzymes  which  must  be  termed  tryptases  are  contained  in  the 
leucocytes  and,  as  Jochmann  showed,  not  only  in  leucemic,  but 
also  in  normal,  leucocytes.  This  occurrence  is  limited,  so  far  as  is  known 
to  man,  monkeys,  and  dogs  (E  r  b  e  n  ,  Munch.  Med.  Wochens.,  1906 
and  1907;  Jochmann  and  Lockemann,  Hofm.  Beitr.,  1908, 
11,  449).  The  tryptase  of  the  leucocytes  is  less  sensitive  to  heat  than 
that  of  the  pancreas;  its  optimal  temperature  is  55°. 

Tryptases  have  also  been  found  in  insects,  protozoa,  sponges,  worms, 
and  molluscs. 

Preparation.  Active  but  impure  solutions  may  be 
obtained  by  extracting  finely-chopped  pancreatic  glands  with 
chloroform- water.  The  best  material  is  pancreatic  juice  obtained 
by  P  a  w  1  o  w  '  s  method  and  activated  by  intestinal  secretion. 

The  purification  of  trypsin  has  been  recently  attempted, 
especially  by  Mays  (H.,  1903,  38,  428  and  1906,  49,  124 
and  188).  He  obtained  a  relatively  very  pure  and  active  prepa- 
ration by  salting-out  the  trypsin  solution  with  sodium  chloride 
and  magnesium  sulphate;  but  his  investigations  indicate  little 
concerning  the  chemical  nature  of  trypsin.  It  is  not  a  nucleo- 
protein  and  does  not  give  the  biuret  reaction  (Mays, 
Schwarzschild). 

In  aqueous  solution,  it  is  very  labile  and  sensitive  to  heat, 
and,  indeed,  more  so  in  alkaline  than  in  neutral  solution.  Sub- 
strate and  decomposition  products  exert  a  considerable  protecting 
action  (Bayliss,  Vernon),  so  that  the  optimal  tem- 
perature is  given  as  40°.  Trypsinogen  is  less  sensitive  to  heat 
than  trypsin.  Acids  denature  it  even  in  low  concentrations. 
In  contrast  to  pepsin,  which  dissolves  in  alkali,  trypsin  is 


38  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

soluble  in  acids.  In  the  electric  field,  it  behaves  as  an  ampho- 
teric  electrolyte,  that  is,  it  migrates  either  to  the  anode  or  to 
the  cathode  according  to  the  reaction  of  the  solution.  It  is 
affected  but  slightly  by  chloroform  or  thymol. 

Pollak  (Hofm.  Beitr.,  1905,  6,  95)  characterises  glu- 
tinase  as  a  separate  enzyme,  owing  to  its  resistivity  towards 
acids.  It  acts  on  glue,  but  not  on  certain  other  proteins. 

On  the  other  hand,  A  s  c  o  1  i  and  N  e  p  p  i  (H.,  1908, 
56,  135)  have  established  the  fact  that  the  specificity  of  glutinase 
towards  glue  is  only  apparent  and  is  due  to  the  different  proteins 
being  influenced  differently  by  activators  and  paralysers 

Er  ep  sin 

See  Cohnheim  (H.,  1901,  33,  651;  1902,  35,  134;  1902, 
36,  13;  1906,  49,  64).  This  enzyme  decomposes  albumoses, 
polypeptides,  peptones  and  protamines  completely  into  amino- 
acids.  With  the  exception  of  casein,  proteins  are  not  attacked, 
but  nucleic  acids  are  decomposed  (Nagayama,  H.,  1904, 
41,  348). 

Occurrence:  in  the  intestinal  juice  and  the  intestinal  mucous 
membrane,  the  latter  being  the  richer  in  the  enzyme.  It  appears,  especi- 
ally after  V  e  r  n  o  n  '  s  investigations  (Journ.  of  Physiol.,  1904,  32,  33) 
and  those  ofAbderhalden,  to  be  the  most  widely  distributed 
proteolytic  enzyme;  it  has  been  detected  in  nearly  all  the  animals 
examined  for  its  presence. 

Preparation.  According  to  Cohnheim,  it  can 
be  prepared  from  the  pressed  juice  of  the  intestinal  mucous 
membrane;  also  extraction  of  the  latter  with  glycerol  or  water 
yields  very  active  solutions.  From  the  aqueous  solution,  the 
enzyme  is  salted  out  with  ammonium  sulphate.  Pressed  yeast 
juice  likewise  contains  much  erepsin  (Abderhalden). 

The  optimal  temperature  is  about  38°  (in  alkaline  solution). 

PROTEOLYTIC   ENZYMES   OF  PLANTS 

Proteinases  is  the  name  given  to  those  enzymes  which 
break  down  the  true  proteins.  The  decomposition  appears  to 
proceed  as  far  as  the  albumoses  and  peptones.  It  is  seldom 
that  proteinases  occur  alone  in  the  organs  of  plants  where 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  39 

proteins  are  decomposed;  they  are  almost  always  accompanied 
by  peptases,  which  correspond  with  the  erepsins  of  the  animal 
body.  Vines  (Annals  of  Bot.,  1904,  18,  289;  1905,  19,  149, 
171;  1906,  20,  113;  1908,  22,  103;  1909,  23,  1)  to  whom  we  owe 
a  thorough  investigation  of  the  vegetable  proteinases  has,  indeed, 
recently  found  both  enzymes  in  the  seeds  of  Cannabis 
s  a  t  i  v  a  . 

Occurrence.  Proteinases  occur  especially  in  germinating  and 
ungerminated  seeds  and  more  abundantly  in  oil-bearing  than  in  starch- 
containing  seeds — particularly  in  Cannabis  sativa,  Sinapis, 
R  i  c  i  n  u  s  and  L  i  n  u  m  ;  further,  in  certain  juicy  fruits  (F  i  c  u  s 
c  a  r  i  c  a  )  and  leaves  (Agave).  They  are  generally  found  in  insect- 
ivorous plants.  Among  those  which  have  been  thoroughly  investigated 
are 

B  r  o  m  e  1  i  n  in  acid  banana-juice  (Chittenden,  Journ.  of 
Physiol.,  1894,  15,  249);  according  to  C  aid  well  (Bot.  Gaz.,  1905, 
39,  407),  this  consists  of  two  components,  a  pepsinase  and  a  tryptase;  and 

Papain  or  papayotin  in  the  juice  of  the  fruit,  leaves  and 
stem  of  the  papaw  tree  (Carica  papaya).  These  two  enzymes 
are  very  closely  allied  if  not  absolutely  identical.  Animal  proteins,  such 
as  fibrin,  are  also  hydrolysed  by  bromelin  (Chittenden).  From 
the  mixture  of  proteolytic  enzymes  in  oily  seeds,  Vines  isolated  a 
fibrin-digesting  proteinase.  A  similar  action  is  exhibited  by  the  papain  of 
Carica  which  readily  digests  fibrin.  After  digestion  of  fibrin  with 
Carica-  sap  and  with  an  enzyme  similar  to  papain  and  obtained  from 
Bacillus  fluorescens  1  i  q  u  e  f  a  c  i  e  n  s  ,  Emmerling 
(Chem.  Ber.,  1902,  35,  695)  found  in  addition  to  albumoses  and  pep- 
tones, also  various  amino-acids,  such  as  leucine,  tyrosine,  etc.  With 
"papayotin  Merck,"  Kutscher  and  Lohmann  (H.,  1905,  46, 
383)  obtained  analogous  results.  According  to  Abderhalden  and 
Teruuchi,  H.,  1906,  49,  21),  papain  splits  glycyl-1-tyrosine;  this 
also  indicates  the  simultaneous  occurrence  of  proteinases  and  peptases, 
unless,  indeed,  papain  is  regarded  as  a  tryptase  with  a  wider  sphere  of 
action. 

Vegetable  proteases  accompany  malt-diastase  and  taka- 
diastase.  These  proteinases  are  extractable  by  alcohol  in  a 
remarkable  manner  (Vines,  Annals  of  Bot.,  1910,  24,  213). 

The  statements  made  concerning  the  acidity  or  alkalinity 
of  the  medium  in  which  vegetable  proteinases  exhibit  their 
maximal  activity  differ  considerably  (cf .  Reynolds  Green, 
Vines,  Emmerling,  Weis,  and  others) .  This  dis- 


40  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

agreement  depends  partly  on  qualitative  and  quantitative 
differences  between  the  substrates  employed  and  partly  on 
varying  composition  of  the  enzyme  preparations.  The  peptase 
present  will  always  act  best  in  neutral  or  faintly  alkaline  solu- 
tions and  the  proteinases,  on  the  other  hand,  in  acid  ones. 
Proteinases  occur  abundantly  in  fungi  and  they  can  be  extracted 
from  the  mycelia  by  water  or  glycerol. 

As  extracellular  enzymes,  proteinases  occur  in  insectivorous 
plants.  In  these  they  are  liberated  from  zymogens  in  certain 
glands  under  the  stimulative  action  of  nitrogenous  substances. 
With  Nepenthes,  the  enzyme  is  distributed  in  the  anti- 
septic, almost  protein-free  sap  of  the  leaf -pitchers.  This  acid 
sap  decomposes  proteins  not  merely  into  albumoses,  but  also 
into  amino-acids  (Vines,  Annals  of  Bot.,  1897,  11,  563). 
But  Abderhalden  found  that  glycyl-1-tyrosine  is  not 
attacked.  Hence  uncertainty  still  prevails  regarding  the  nature 
of  the  enzyme  or  enzymes  of  Nepenthes.  The  leaf-glands 
of  D  i  o  n  a  e  a  and  D  r  o  s  e  r  a  and  the  leaf -edge  of  P  i  n- 
g  u  i  c  u  1  a  yield  acid,  mucilaginous  secretions  which  attack 
proteins  of  cartilaginous  and  glutinous  tissues.  Extracellular 
proteinases  also  appear  to  be  produced  by  bacteria,  especially 
by  those  species  which  are  capable  of  liquefying  gelatine. 

Another  group  of  fungus-proteinases  consists  of  typical  endo- 
enzymes;  noteworthy  among  these  is  the 

Endot-ryptase  of  yeast  (Hahn  and  Geret). 
So  far  as  is  known,  its  action  extends  to  all  proteins  (gelatine, 
fibrin,  casein,  egg-albumin),  which  it  resolves  into  amino-acids. 
It  cannot  be  extracted  from  yeast  by  water,  and  is  obtainable 
only  byBuchner's  method. 

Unlike  papain,  endotryptase  is  most  active  in  acid  solution. 
Its  temperature-optimum  is  40-45°. 

^-Proteases.  Papayotin,  Hahn  and  G  e  r  e  t '  s 
endotryptase  and  the  enzymes  which  effect  a  partial  decomposition 
of  the  protamines  and  complicated  proteins  in  faintly  acid  solu- 
tion, are  classed  together  by  Takemura  as  ^-proteases. 
The  action  of  pepsin  on  protamines  would  thus  be  attributable 
to  the  presence  of  a  g-protease. 

Peptases  are  those  enzymes  which  decompose  albumoses, 
peptones  and  polypeptides  into  amino-acids.  They  generally 
accompany  the  proteinases  and,  as  has  been  shown  by  Vines 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  41 

(Annals  of  Bot.,  1906,  20,  113),  Abderhalden  and  S  c  h  i  t  - 
tenhelm  (H.,  1906,  49,  26)  and  Euler  (H.,  1907,  61, 
244),  they  occur  abundantly  in  the  seeds  of  lupins,  rape,  peas 
and  maize.  Abderhalden  and  his  collaborators  found  it 
also  in  pressed  yeast  juice. 

According  to  certain  investigators,  among  them  W  e  i  s 
(H.,  1900,  31,  79)  and  Vines  (Annals  of  Bot.,  1903,  17,  237 
and  1904, 18,  289),  the  most  rapid  hydrolysis  takes  place  in  faintly 
acid  solution,  whilst  others,  e.g.,  W  i  n  d  i  s  c  h  ,  assert  that  it 
occurs  in  a  slightly  alkaline  medium. 

NUCLEASES 

The  decomposition  of  the  nucleo-proteins  begins  with  their 
resolution  into  nucleic  acids  and  protein-components,  a  reaction 
which  is  brought  about  by  pepsin  or  trypsin.  The  further 
division  of  the  nucleic  acids  is  not,  however,  produced  by  the 
true  proteinases  (Sachs;  Abderhalden  and  Schitten- 
helm,  H.,  1906,  47,  452),  but  by  a  special  group  of  enzymes, 
the  nucleases.  These,  therefore,  resolve  the  nucleic  acids 
into  their  constituents,  purine  or  pyrimidine  bases  (adenine, 
guanine,  cytosine,  thymin),  pentoses,  and  phosphoric  acid. 

That  the  decomposition  of  nuclein,  and  the  digestion  of 
Buchner's  pressed  yeast  juice  are  enzymic  in  character 
has  been  shown  by  Salkowski  (H.,  1889,  13,  506)  and 
H  a  h  n  and  G  e  r  e  t  (Chem.  Ber.,  1898,  31,  2335)  respectively, 
and  more  recent  researches  prove  that  a  number  of  enzymes 
act  on  the  nucleic  acids.  These  enzymes  may  be  divided  into 
a  number  of  groups  (cf.  B  .  B  1  o  c  h  ,  Biochem.  Centralbl., 
1907,  5,  561). 

1.  Nuclease,    which  liberates  the  phosphoric  acid  from 

the  nucleic  acid  molecule. 

2.  Hydrolytic  enzymes,   which  split  off  ammonia  from  the 

aminopurines  and  replace  the  amino-group  by  hydroxyl. 

3.  Oxidising   enzymes   which  oxidise  hydroxypurines  to  uric 

acid. 

4.  Uric  acid  oxydases. 

According  to  Sachs  (H.,  1905,  46,  337),  pure  trypsin  has 
no  action  at  all  on  nucleic  acids.  Decomposition  of  nucleic 


42  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

acids  takes  place  only  in  fresh  pancreas  extracts,  which  exert 
but  slight  proteolytic  action. 

Occurrence.  Very  frequent  in  animal  organs,  e.g.,  in  the 
spleen,  liver  (Jones),  and  in  the  pancreatic  and  thymus  glands 
(Kutscher,  H.,  1901,  34,  114).  It  performs  an  important  function 
in  many  organs  of  plants  —  more  particularly  in  germinating  seeds  —  in 
synthesizing  and  decomposing  nucleo-proteins  (Z  a  1  e  s  k  i  ,  Bot.  Ber., 
1907,  25,  349).  Worthy-  of  note  also  are  the  nucleases  of  higher  (K  i  k- 
k  o  j  i  ,  H.,  1907,  51,  201)  and  lower  fungi  (I  w  a  n  o  f  f  ,  H.,  1903,  39,  31), 
especially  those  of  yeast. 

P.  A.  Levene  and  Medigreceanu  (Journ.  of 
Biol.  Chem.,  1911,  9,  389)  have  recently  proposed  the  following 
classification  and  nomenclature. 

Nucleinases  resolve  the  nucleic  acid  molecule  into 
nucleotides. 

Nucleotidases  decompose  nucleotides  into  phos- 
phoric acid  and  a  carbohydrate-base  complex  (nucleoside)  . 

Nucleosidases  hydrolyse  nucleosides  into  ribose  and 
purine  bases. 

Arginase   (Kossel  and  Dak  in,   H.,  1904,  41,  321) 

This  enzyme  hydrolyses  arginine  specifically  into  urea  and 
ornithine  according  to  the  following  equation: 


NH  NH2 

+NH2  •  CH2  •  CH2  •  CH2  •  CH  •  COOH. 

I 
NH2 

Whilst  trypsin  hydrolysis  may  be  indicated  by  the  scheme: 

C—  CO-j-NH-C, 
the  action  of  arginase  is  represented  by 


Occurrence.     In   the   liver,    kidneys,    thymus,    and   intestinal 
mucous  membrane  of  the  calf  and  also  in  the  muscles  of  the  dog  and 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  43 

the  lymphatic  glands  of  cattle.    Shiga    (H.,   1904,  42,  502)  found 
arginase  also  in  yeast. 

It  can  be  extracted  from  the  organs  by  means  of  water. 

Ur ease 

This  enzyme  decomposes  urea  into  carbon  dioxide  and 
ammonia. 

Occurrence.  According  to  L  e  u  b  e  (Virch.  Arch.,  1885,  100, 
540), urease occurs  inMicrococcus  ureae,  from  which  it  passes 
readily  into  the  surrounding  liquid.  In  cystitic  urine  the  enzyme  occurs 
only  when  fungi  capable  of  decomposing  urea  are  present. 

Lea  (Journ.  of  Physiol.,  1885,  6,  136)  has  attempted  to 
prepare  urease  in  a  pure  state;  by  treatment  of  Micro- 
coccus  ureae  with  alcohol,  extraction  of  the  precipitate 
with  water  and  repeated  reprecipitation  with  alcohol,  a  powder 
is  obtained  which  yields  a  clear  aqueous  solution  but  contains 
protein.  As  later  work  has  shown,  urease  is  tenaciously  retained 
by  living  protoplasm.  Moll  (Hofm.  Beitr.,  1902,  2,  344) 
also  obtained  the  enzyme  in  a  similar  manner  and  from  the 
same  material;  it  readily  undergoes  decomposition.  Schit- 
t  e  n  h  e  1  m  obtained  an  enzyme-solution  (free  from  purine- 
bodies)  from  the  kidneys  and  K  i  k  k  o  j  i  found  urease  in  a 
pileate  fungus. 

AMIDASES    (DESAMIDASES) 

Decomposition  of  the  proteins  by  trypsin  and  erepsin  ceases 
when  the  amino-acids  are  reached,  but  the  desamidases  attack 
the  latter,  decomposing  them  into  ammonia  and  hydroxy-acids. 
Such  simple  desamination,  as  was  discovered  byH.  Prings- 
heim  (Biochem.  Z,  1908,  12,  15),  takes  place  by  virtue  of 
an  enzyme  present  in  acetone-yeast.  Similar  action  is  exhibited 
by  permanent  preparations  of  Aspergillus  niger  (Shi- 
bat  a,  Hofm.  Beitr.,  1904,  5,  384).  It  has  been  shown  by 
Pringsheim,  Abderhalden  and  Schittenhelm 
that  the  amidases  of  yeast  do  not  pass  into  the  press-juice.  Of 
biological  importance  is  the  detection  of  amidases  in  higher 
plants  (Butkewitsch,  H.,  1909,  63,  103;  cf.  Kiesel, 
H.,  1910,  65,  283). 


44  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

The  so-called  "  alcoholic  fermentation  of  the  ammo-acids  " 
—  discovered  by  F  ,  E  h  r  1  i  c  h  —  consists  of  a  combination 
of  two  reactions,  namely  the  desamination  of  the  amino-acids 
to  the  corresponding  hydroxy-acids  and  the  subsequent  loss  of 
carbon  dioxide  from  the  latter;  the  total  reaction  can  hence 
be  formulated  thus: 


Reactions  similar  in  principle  to  those  brought  about  by 
amidases  are  caused  by  the  enzymes  known  as  guanase 
and  a  d  e  n  a  s  e  .  These  convert  guanine  and  adenine  into  the 
corresponding  hydroxy-derivatives  : 

N  =  C—  NH2  N  =  C—  OH 

II  II 

CH   C—  NH,  +  H20  =      CHC—  NH, 

II      II  >CH  ||      ||  >CH  +  NH3; 

N—  C  -  W  N—  C  -  W 

Adenine  Hypoxanthine 

and 

NH—  CO 

NH2-C        C—  NHX       +H20  =  OH-C 

II         II  >CH  ||        ||  CH+NH3 

N  -  C  --  N^ 

Guanine  Xanthine 

Concerning  the  individuality  and  mode  of  action  of  these 
enzymes  of  the  purine-bases,  various  views  have  been  expressed 
by  the  different  investigators  who  have  studied  them  (Schitten- 
helm,  H.,  1904,  42,  251;  1905,  43,  228;  45,  121,  152;  46, 
354;  Jones  and  his  collaborators,  H.,  1904,  42,  35,  343;  1905, 
44,  1;  45,  84;  1906,  48,  110;  and  also  Burian,  H.,  1905, 
43,  494)  .  Even  if  Schittenhelm's  assumption  that  the 
two  enzymes  are  identical  is  not  correct,  they  are  certainly  very 
similar. 

Occurrence.  Guanase  and  adenase  have  been  found  in  the 
pancreas,  spleen,  lungs,  and  liver  of  the  child,  pig  (not  in  the  spleen), 
cattle,  and  also  in  numerous  other  organs. 

Preparation.  (Schittenhelm,  H.,  1904,  42, 
251).  One  or  two  spleens  are  extracted  with  about  2  litres  of 
water  for  12  hours  and  the  enzyme  then  precipitated  with 


SPECIAL  CHEMISTRY  OF   THE  ENZYMES  45 

ammonium  sulphate.  The  precipitate  is  dissolved  in  water 
and  freed  from  ammonium  sulphate  by  dialysis. 

Ni  t  r  ilase 

According  to  Rosenthaler  (Biochem.  Z.,  1909,  19, 
186;  1910,  28,  408),  the  decomposition  of  amygdalin  is  effected 
by  three  enzymes — an  amygdalase,  a  ^-glucosidase  and  an 
hydroxynitrilase.  The  last  of  these  exerts  a  catalytic  influence 
on  the  reaction. 

OH 

C6H5-CH<f        +H20  =  C6H5-CHO+HCN, 
CN 

and,  in  Rosenthaler's  opinion,  its  action  is  solely 
hydrolytic,  i.e.,  it  can  be  separated  from  the  synthesising  con- 
stituent of  emulsin;  in  Chapter  VII  of  this  book,  hydroxynitrilase 
(oxynitrilase)  will  be  considered,  together  with  the  corresponding 
synthesising  enzyme,  in  greater  detail. 

COAGULATING  ENZYMES 

C  h  y  m  o  s  i  n  and  parachymosin  are  those  enzymes 
which  effect  the  clotting  of  milk,  the  casein  of  the  latter  being 
changed  in  some  way,  as  yet  unknown.  The  two  enzymes  are 
distinguished  according  to  their  place  of  origin.  Chymosin 
which  has  been  long  known  and  is  the  enzyme  of  the  calf's 
stomach,  is,  according  to  Bang  (Deutsch.  med.  Wochens.,  1899, 
and  Pfliig.,  Arch.,  1900,  79,  425),  to  be  distinguished  from  the 
coagulating  constituent  of  the  human  stomach,  namely,  para- 
chymosin. The  chemistry  of  rennet-action  is  still  not 
clear.  With  solutions  containing  pure  casein  and  also  rennet  in  as 
pure  a  state  as  possible,  the  clear  whey  obtained  after  coagulation 
contains  a  very  small  proportion  of  a  protein — whey-albumin — 
with  only  13-2%  of  nitrogen.  The  bulk  of  the  casein  is  precip- 
itated as  a  substance,  paracasein,  very  similar  to  casein 
itself.  Whether  decomposition  of  the  casein  occurs  is  still 
uncertain  (cf .  Hammarsten,  Text-book  of  Physiological 
Chemistry,  4th  Edition,  1906,  442). 

Occurrence.  In  the  gastric  juice,  the  gastric  mucous  membrane, 
the  pancreatic  juice,  the  placenta,  and  certain  other  organs  of  many 


46  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

different  anLnals  The  enzyme  often  occurs  in  an  inactive  form  as  the 
so-called  prochymosin  (Edmunds,  Journ.  of  Physiol.,  1895, 
19,  466;  Vernon,  Journ.  of  Physiol.,  1901,  27,  174),  owing  mainly 
to  the  lack  of  an  activator. 

The  activation  of  prochymosin  can  be  effected  instantly  by 
an  acid  and,  after  activation,  the  rennet  is  active  in  both 
neutral  and  alkaline  solutions. 

Preparation.  (Hammarsten,  H.,  1908,  56,  18). 
This  author  has  recently  described  the  preparation  of  chymosin 
from  the  stomach  of  the  calf,  horse,  hen  and  pike.  The  first- 
named  material  is  treated  as  follows: 

The  innermost  coat  of  the  stomach  is  separated  from  the 
intestines  and  from  the  three  other  stomachs,  cut  along  the 
small  curvature  and  freed  from  contents  by  rinsing  out  with 
water.  The  pylorus  portion  is  then  cut  away  together  with  at 
least  3-5  c.m.  of  the  large  fold  of  the  fundus  portion.  (The 
pylorus  part  yields  a  more  mucilaginous  infusion  and  is,  at  the 
same  time,  less  rich  in  enzymes,  than  is  the  fundus.)  The 
remainder  of  the  stomach  is  stretched  out  and  thoroughly  washed. 
The  glandular  layer  is  then  scraped  from  the  two  sides  of  a  fold 
with  the  edge  of  a  watch-glass,  weighed  and  introduced  into 
0-1-0-2%  hydrochloric  acid,  10-20  c.c.  of  the  latter  being  taken 
per  grm.  of  the  glandular  matter.  The  acid  is  allowed  to  act, 
with  repeated  shaking,  for  12-24  hours  at  a  low  temperature — 
somewhat  above  0° — the  liquid  being  then  filtered.  The  infusion 
is  neutralised  and,  if  found  to  exert  a  vigorous  coagulating  action, 
is  precipitated  with  magnesium  carbonate. 

One  grm.  of  magnesium  carbonate  is  added  to  each  100 
c.c.  of  the  extract,  which  is  shaken  and  quickly  filtered;  it 
can  then  be  tested  for  the  presence  of  pepsin  (see  Appendix: 
Practical  Methods).  If  the  filtrate  still  contains  much  of  this 
enzyme,  it  is  again  treated  with  magnesium  carbonate,  and 
this  procedure  is  repeated  until  a  filtrate  is  obtained  which 
readily  causes  clotting  of  milk  but  has  only  a  slight  action  on 
fibrin. 

Another  process  which  Hammarsten  gave  for  the 
preparation  of  chymosin  consists  in  freeing  it  from  the  greater 
part  of  the  pepsin  by  means  of  magnesium  carbonate  and  pre- 
cipitating the  chymosin  with  lead  acetate.  The  lead  is  removed 
from  the  precipitate  by  sulphuric  acid  and  the  acid  filtrate  shaken 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  47 

with  an  alcoholic  solution  of  cholesterol  or  stearic  acid  and  a 
little  ether,  so  that  the  precipitated  cholesterol  or  stearic  acid 
carries  down  the  enzyme  with  it.  If  now  the  precipitate  is 
shaken  with  water  and  freed  from  the  precipitant  by  treatment 
with  ether,  a  moderately  pure  chymosin  solution  is  obtained. 
The  purest  clotting  enzyme  obtained  in  this  way  did  not  give 
the  ordinary  reactions  for  proteins. 

Two  different  views  are  held  as  to  the  identity  of  chymosin 
and  pepsin.  According  to  the  one,  which  has  been  advanced 
by  Pawlow  (H.,  1904,  42,  415),  Saw  j  alow  (H.,  1905, 
46,307),  Sawitsch  (H.,  1908,  55,  84)  and  Gewin  (H., 
1907,  54,  32),  both  actions  result  from  one  and  the  same  enzyme, 
S  a  w  j  a  1  o  w  and  Gewin  regarding  the  clotting  of  milk  as 
the  commencement  of  pepsin-digestion.  On  the  other  hand, 
Nencki  and  Sieber  (H.,  1901,  32,  291),  Pekelharing 
(H.,  1902,  35,  8),  Schmidt-Nielsen  (H.,  1906,  48,  92), 
Taylor  (Journ.  of  Biol.  Chem.,  1909,  5,  399),  and  especially 
Hammarsten  (H.,  1908,  56,  18)  are  of  opinion  that  the 
two  enzymes  are  not  identical  but  of  different  kinds. 

In  addition  to  the  cholesterol  method  described  above, 
Hammarsten  has  recently  given  a  second  method  allowing 
of  the  separation  of  chymosin-action  from  that  of  pepsin.  In 
principle  this  method  consists  in  heating  the  acid  enzyme- solu- 
tion to  40°  or  a  rather  higher  temperature.  In  this  way  the 
calf-chymosin  is  destroyed  more  rapidly  than  the  pepsin,  so 
that  after  some  time  a  solution  is  obtained  which  no  longer 
exerts  a  clotting  action,  but  still  digests  proteins  ( 1  o  c  .  c  i  t  .  , 
p.  61). 

Still  more  recently  (H.,  1911,  74,  142),  Hammarsten  has 
succeeded  in  preparing  pepsin-free  chymosin  solutions  by  mixing 
an  acid  infusion  of  calf's  stomach  and  a  neutral  alkali  caseinate 
solution  in  such  proportions  that  the  casein  at  first  separating 
just  redissolves.  To  the  acid  casein  solution  thus  obtained, 
decinormal  sodium  hydroxide  solution  is  added  in  sufficient 
quantity  to  produce  an  abundant  precipitation  of  casein  and  to 
allow  of  ready  filtration  while  the  reaction  still  remains  strongly 
acid.  Both  the  enzymes  are  carried  down  by  the  precipitated 
casein,  but  the  pepsin  in  much  larger  quantity  than  the  chymosin. 
The  filtrate  therefore  contains  a  relatively  high  proportion  of 
chymosin. 


48  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Chymosin  exhibits  remarkable  activity,  one  part  of  the 
mucous  membrane  of  the  calf's  stomach  being  sufficient  to 
clot  250,000  parts  of  milk.  Purified  chymosin  coagulates 
24,000,000  (Hammarsten)  or  30,000,000  (  F  u  1  d  )  times 
its  weight  of  milk. 

Chymosin  causes  clotting  only  in  acid  solutions;  hydro- 
chloric acid  is  the  most  favourable  to  its  action  and  after  this 
come  nitric,  lactic,  acetic,  sulphuric  and  phosphoric  acids.  Its 
optimum  temperature  is  37-39°. 

Chymosin  is  injured  by  chloroform  (Benjamin),  but 
not  by  hydrocyanic  acid  (  F  u  1  d  and  S  p  i  r  o  ). 

Milk-clotting  Enzyme  in  Plants    (Cynarase) 

A  large  number  of  plants,  such  as  Pinguicula  v  u  1  - 
garis,  Galium  verum,  artichokes,  etc.,  possess  the 
property  of  rendering  milk  ropy. 

Whether  the  chemical  change  underlying  this  coagulation 
is  or  is  not  the  same  as  that  produced  by  the  action  of  chymosin, 
is  unknown. 

The  vegetable  chymases  must,  however,  be  quite  different 
from  the  animal  enzymes.  According  to  C  h  o  d  a  t  and 
Rouge  (Centralbl.  f.  Bakt.,  1906,  II,  16,  1),  the  syko- 
c  h  y  m  a  s  e  from  Ficus  carica  investigated  by  them 
acts  in  absence  of  calcium  salts.  Its  optimal  temperature  is 
75-80°. 

Further,  as  has  been  shown  by  Bruschi  (Atti  Real. 
Accad.  Lincei,  1907,  [V],  16,  ii,  360)  and  especially  by  G  e  r  b  e  r 
(C.  R.,  1907,  145,  689;  1908,  146,  1111;  147,  601,  1320;  1909, 
148,  497,  992;  149,  137,  737;  1910,  150,  1202,  1357),  the  phyto- 
chymases  exhibit  great  differences  among  themselves.  G  e  r  b  e  r 
distinguishes  these  enzymes  according  to  the  amounts  of  lime 
they  require  for  their  action,  and  he  has  further  shown  that 
some  phyto-chymases  coagulate  raw  milk,  while  others  coagulate 
boiled  milk  the  more  readily. 

Occurrence.  In  addition  to  the  plants  mentioned  above,  the 
following  also  exert  a  coagulating  action  on  milk:  Lolium  perenne, 
Anthriscus  vulgaris,  Plantago  lanceolata,  La- 
mium  amplexicaule  and  hybridum,  Philadelphus 
c  oronarius  ,  Geranium  m  o  11  e  ,  C  ap  s  e  11  a  bursa  pas- 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  49 

tor  is,  Ranunculus  bulbosus,Medicago  lupulina, 
Centaurea  scabiosa,  etc.  Reynolds  Green  (Proc.  Roy. 
Soc.,  1890,  48,  370)  found  the  enzyme  in  'the  germinating  seeds  of 
Ricinus  communisin  the  form  of  a  zymogen,  which  is  activated 
by  dilute  acids.  This  enzyme  is  also  contained  in  numerous  other 
seeds,  for  example,  in  those  of  Datura,  Pisum,  and  L  u  p  i  n  u  s 
hirsutus.  Weis  (H.,  1900,  31,  79)  found  a  clotting  enzyme  in 
malt.  The  coagulating  action  of  the  fig,  F  i  c  u  s  c  a  r  i  c  a  ,  on  milk 
was  known  to  the  ancient  Greeks.  See  also  G  e  r  b  e  r  (C.  R.,  1909, 
148,  992). 

Lastly,  coagulating  enzymes  have  also  been  detected  in  many  lower 
fungi,  among  others  Fuligo  varians.  C.  Gerber  has  recently 
examined  86  species  and  sub-species  of  Basidiomycetes,  and 
has  found  them  to  contain,  in  general,  an  "oxyphile  "  and  a  "calciphile" 
coagulating  enzyme. 

Thrombin,    Fibrin-ferment  (Alexander   Schmidt,    1872) 

Thrombin  causes  blood  to  coagulate  by  converting  dissolved 
fibrinogen  into  insoluble  fibrin. 

The  change  taking  place  is  probably  as  follows:  When  the 
blood  leaves  the  body,  one  of  its  constituents  (possibly  the 
leucocytes)  gives  rise  to  a  pro-enzyme,  which  is  converted  into 
the  active  enzyme  under  the  influence  of  the  calcium  salts. 
This  enzyme,  without  further  action  of  calcium  salts,  then  trans- 
forms the  fibrinogen  into  insoluble  fibrin. 

It  is  best  prepared  from  blood-serum  or  defibrinated  blood 
by  precipitation  with  15-20  volumes  of  alcohol,  which  separates 
the  proteins  at  the  same  time.  If  the  precipitate  is  then  extracted 
with  water,  part  of  the  protein  remains  undissolved,  whilst  the 
thrombin  passes  into  solution.  According  to  Hammar- 
s  t  e  n '  s  method  (Pflug.  Arch.,  1878,  18,  38)  the  globulins 
are  first  precipitated  by  magnesium  sulphate;  the  liquid  is  then 
diluted  with  water  and  sodium  hydroxide  solution  added  so  as  to 
precipitate  magnesium  hydroxide,  which  is  accompanied  by  a  con- 
siderable amount  of  adsorbed  fibrin-ferment.  Pekelharing 
dialyses  the  filtrate  from  the  precipitate  given  by  magnesium 
sulphate.  From  the  muscles  of  birds,  F  u  1  d  obtained  throm- 
bin by  extraction  with  0-8%  sodium  chloride  solution. 

According  to  S  h  i  g  e  j  i  and  H  i  g  u  c  h  i ,  the  placenta 
contains  a  fibrin-enzyme,  which  can  be  extracted  by  means  of 
water  or  physiological  salt  solution. 


50  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Pekelharing  regards  thrombin  as  the  lime  compound  of 
pro-thrombin,  the  nucleoprotein  which  occurs  in  blood-plasma 
and  which  he  attempted  to  prepare  in  a  pure  condition  (Verh. 
d.  k.  Akad.  v.  Wetenschappen  te  Amsterdam,  1892,  II,  1,  No.  3; 
and  Zentralbl.  f.  PhysioL,  1895,  9,  102).  The  existence  of  this 
nucleoprotein  in  blood-serum  has  been  established,  but  its  com- 
position has  not  yet  been  investigated  since  the  quantity  of  it 
in  blood  is  very  small.  In  a  concentrated  solution  of  the  enzyme, 
which  contained  0-417%  of  organic  matter  and  0-166%  of  inor- 
ganic matter,  Hammarsten  found  only  0  •  005%  of  nuclein. 
The  experimental  results  obtained  by  Huiskamp  (H., 
1901,  32,  145),  led  this  investigator  to  dispute  Pekelharing 's 
views.  Huiskamp  found  that,  in  presence  of  calcium  salts, 
both  the  nucleohistone  and  the  other  nucleoprotein  of  the  thymus 
glands — two  essentially  different  substances — acted  on  fibrin- 
ogen  in  the  same  way  as  thrombin;  for  the  calcium  nucleo- 
protein he  gave  the  following  percentage  composition :  C,  49  •  82  ; 
H,  7-29;  N,  15-81;  P,  0-954;  S,  1-188  and  Ca,  1-337.  The 
question  whether  the  protein  itself  is  to  be  regarded  as  the 
fibrin-enzyme  or  whether  its  action  on  the  formation  of  fibrin 
is  due  to  an  admixture  with  another  substance  is  left  undecided 
by  Hammarsten  (Ergeb.  der  Physiol.,  1902,  1,  i,  339). 

The  optimal  temperature  for  thrombin  is  40°. 

For  the  chemistry  of  the  coagulation  of  blood  see  Mora- 
witz,  Hofm.  Beitr.,  1903,  4,  381;  1904,  5,  133;  L.  Loeb, 
Biochem.  Zentralbl.,  1907,  6,  829,  889;  Pekelharing, 
Biochem.  Z.,  1908,  11,  1. 

ENZYMES   OF  FERMENTATION 

Fermentation  is  not  a  chemically  definite  conception;  by  it 
are  understood  those  processes  which  are  brought  about  by 
lower  organisms  and  the  extent  of  which  is  great  compared 
with  the  mass  of  the  organisms  taking  part. 

From  a  chemical  point  of  view,  fermentation  enzymes  can 
be  contrasted  with  the  hydrolytic  enzymes  in  so  far  as  fermen- 
tation reactions  consist  of  pure  decompositions  and  take  place 
without  any  other  substance,  such  as  water  or  oxygen,  being 
taken  up;  the  best-known  example  is  the  alcoholic  fermentation 
of  the  hexoses  which  is  effected  by  zymase.  On  the  other  hand, 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  51 

the  enzyme  of  the  acetic  acid  fermentation  is  regarded  as  belonging 
not  to  the  same  group  but  to  the  oxydases.  The  reactions  under- 
lying other  fermentations  have  been  investigated  chemically 
only  in  very  recent  times  and  attention  must  be  drawn  especially 
to  the  work  of  Buchner  and  Meisenheimer;  the 
existence  of  the  enzymes  which  presumably  take  part  in  these 
fermentations  has  not  yet  been  proved.  The  view  that  fermen- 
tations in  general  are  to  be  referred  to  enzyme  actions  is  a  con- 
sequence of  the  discovery  of  E.  Buchner,  who,  in  1897, 
succeeded  in  producing  alcoholic  fermentation  in  a  pressed  yeast 
juice  free  from  cells  and  hence  in  showing  that  this  fermentation 
is  not  dependent  on  the  action  of  the  living  yeast. 


Enzymes    of    Alcoholic    Fermentation 

The  term  zymase,  in  its  wider  sense,  is  used  to  indicate  the 
sum-total  of  the  enzymes  which  bring  about  the  decomposition 
of  certain  of  the  hexoses  in  the  sense  of  the  following  equation: 

C6H1206  =  2C2H5  •  OH+2C02. 

d-Hexose.         Ethyl  alcohol. 

As  was  assumed  by  Buchner  and  Meisenheimer 
and  by  W  o  h  1 ,  this  chemical  change  is  to  be  regarded  as  taking 
place  in  several  separate  stages.  It  was  formerly  thought  that 
glucose  gives  rise  to  lactic  acid  under  the  action  of  an  enzyme 
to  which  the  name  zymase  was  then  applied  in  a  more  restricted 
sense.  Buchner  and  Meisenheimer  have,  indeed, 
detected  the  formation  of  small  quantities  of  lactic  acid  during 
fermentation,  but  S  1  a  t  o  r  (Journ.  Chem.  Soc.,  1906,  89,  128) 
has  shown  that  lactic  acid,  which  is  fermented  with  extreme 
slowness,  can  be  only  a  bye  -product  and  not  an  inter- 
mediate product  of  fermentation.  It  is  now  assumed  that 
compounds  allied  to  lactic  acid  are  formed  at  an  intermediate 
stage  of  the  fermentation,  which  possibly  passes  through  the 
following  stages: 


52  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Methylglyoxal 

CHO  CHO          CHO          CHO        H-CO2H  Formic   CO2 

CH-OH  C-OH        CO  CO  CHO  Acetaide— >  CH2-OH 

CH-OH       HCH         -»CH2  CH3          CH3  CH3 

|  ~~OH-^|  4-1  ->- 

CH-OH  CH-OH      CH-OH     CHO  CHO      CHO 

CH-OH  CH-OH      CH-OH     CH-OH~OH  ""C-OH^CO 

CH2-OH  CH2-OH    CH2-OH    CH2-OH  CH2        CH3 

Glucose         .  Glyceraldehyde  Methylglyoxal 

There  is  also  uncertainty  concerning  the  occurrence  of  methyl- 
glyoxal,  which  cannot  be  fermented  by  pressed  yeast  juice. 
Boysen-Jensen  (Bot.  Ber.,  1908,  26,  666;  also  Dis- 
sertation, Copenhagen,  1910)  supposes  the  intermediate  product 
to  be  dihydroxyacetone,  an  isomeride  of  glyceraldehyde,  but  it 
cannot  be  said  that  this  has  been  proved  to  be  the  case;  this 
assumption  is,  however,  rendered  probable  by  the  fact  that 
dihydroxyacetone  is  fermented  readily  and  glyceraldehyde  only 
slowly. 

It  is  worthy  of  note  that  the  transformation  of  glucose  into 
alcohol + carbon  dioxide  can  be  effected  by  purely  chemical 
means,  the  various  reactions  requiring,  however,  different  catalysts: 

Glucose— ^lactic  acid  (alkali  as  catalyst). 

Lactic  acid— >acetaldehyde+ formic  acid  (sulphuric  acid  as 
catalyst) . 

Acetaldehyde-f  formic  acid— »alcohol+ carbon  dioxide  (rhodium 
as  catalyst). 

(Buchner,  Meisenheimer  and  S  c  h  a  d  e  ,  Chem. 
Ber.,  1906,  39,  4217;  Schade,  Zeitschr.  f.  physikal.  Chem., 
1906,  57,  1.)  Further,  the  author's  investigations  (Arkiv 
for  Kemi,  1911,  4,  No.  8)  show  that,  in  ultra-violet  light,  lactic 
acid  undergoes  decomposition  into  alcohol  and  carbon  dioxide. 

According  to  a  new  and  interesting  investigation  by  Franzen 
and  Steppuhn  (Chem.  Ber.,  1911,  44,  2915),  formic  acid  is 
fermented  as  well  as  formed  by  living  yeast  and  must  hence  be 
taken  into  account  as  an  intermediate  product. 

As  substrate  for  fermentation,  mannose,  galactose,  or  fructose 
may  be  used  instead  of  glucose.  Glucose  and  fructose  exhibit 
no  difference  in  their  velocity  of  decomposition,  and  in  the  case 
of  mannose  there  is  only  a  slight  deviation.  Galactose,  on  the 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  53 

other  hand,  is  fermented  only  by  certain  species  of  yeast,  including 
bottom  fermentation  beer-yeasts,  and  by  their  pressed  juices; 
the  fermentation  takes  place  far  more  slowly  than  that  of  glucose 
(E.  Fischer  and  Thierfelder,  Chem.  Ber.,  1894, 
27,2031;  E.  F.  Armstrong,  Proc.  Roy.  Soc.,  B,  1905, 
76,  600;  SI  at  or,  Journ.  Chem.  Soc.,  1908,  93,  217).  As, 
however,  has  been  shown  by  S  1  a  t  o  r  and  by  Harden 
and  Norris  (Proc.  Roy.  Soc.,  1910,  82,  645),  the  capacity 
of  yeasts  for  fermenting  galactose  can  be  increased  by  culti- 
vating them  in  solutions  containing  this  sugar.  Mention  must 
also  be  made  of  S  1  a  t  o  r  '  s  view  that  the  different  hexoses 
are  attacked  by  different  enzymes:  glucose  and  fructose  by 
gluco-zymase,  mannose  by  manno-zymase  and  galactose  by 
galacto-zymase. 

The  mechanism  of  alcoholic  fermentation  is  considerably  less 
simple  than  was  formerly  supposed,  a  number  of  enzymes  and 
subsidiary  substances  taking  part  in  the  formation  of  alcohol 
and  carbon  dioxide. 

The  first  separation  of  zymase  into  a  "  zymase  in  a  restricted 
sense  "  and  a  lactacidase  must  be  given  up,  since  the  formation 
of  lactic  acid  as  an  intermediate  product  has  been  shown  to  be 
improbable.  And  a  special  enzyme  has  now  to  be  assumed 
for  each  of  the  changes  indicated  in  the  above  scheme  of  reactions. 

Of  great  importance  for  the  elucidation  of  the  nature  of 
fermentation  is  Harden  and  Young's  discovery  of  the 
co-enzyme  of  zymase.  By  filtration  through  a  film  of 
gelatine  under  a  pressure  of  50  atmospheres,  pressed  yeast  juice 
can  be  divided  into  a  filtrate  and  a  residue,  which  are  separately 
inactive  towards  sugar  but  produce  fermentation  when  again 
mixed  (Harden  and  Young,  Proceedings  of  the  Physiol. 
Soc.,  Nov.  12,  1904,  see  Journ.  of  Physiol.,  1904,  32,  i;  Proc.  Roy. 
Soc.,  B,  1906,  77,  405). 

The  substance  in  the  dialysate  resists  boiling  (thermostable) 
and  undergoes  hydrolytic  decomposition  and  hence  destruction 
by  enzymes  (lipases)  of  the  yeast  juice. 

On  the  other  hand,  the  zymase  itself — which  does  not  traverse 
the  gelatine  filter — is  destroyed  when  heated  and  is  presumably 
a  protein  substance,  being  attacked  by  the  proteinases  or  pro- 
teases of  the  yeast  juice.  From  this  attack  it  is  protected  by  a 
thermostable  substance — a ntiprotease  (Buchner  and 


54  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Haehn,  Biochem.  Z.,  1910,  26,  171).  This  antiprotease  is, 
like  the  co-enzyme,  destroyed  by  lipase,  but  is  more  stable  than 
the  co-enzyme  towards  hydrolytic  agents  and  towards  heat. 

Both  enzyme  and  co-enzyme  are  precipitated  from  the  yeast 
juice  by  acetone,  but  the  latter  less  readily  than  the  former, 
so  that  a  certain  degree  of  separation  can  be  attained  by  frac- 
tional precipitation  (  B  u  c  h  n  e  r  and  Duchacek,  Bio- 
chem. Z.,  1909,  15,  221). 

Alcoholic  fermentation  by  means  of  pressed  yeast  juice  is 
facilitated  by  the  addition  of  a  phosphate.  Harden  and 
Young  (Proc.  Roy.  Soc.,  B,  1906,  77,  405)  have  made  the 
important  discovery  that,  during  the  period  of  enhanced  fer- 
mentation, the  amount  of  carbon  dioxide  produced  exceeds  that 
which  would  have  been  formed  in  the  absence  of  phosphate 
by  a  quantity  exactly  equivalent  to  the  phosphate  added  — 
C02  :  R2/HP04. 

These  two  investigators  consider  that  the  phosphate  reacts 
with  the  hexose  in  the  yeast  juice  in  the  following  manner: 


(1) 

+2H20+C6H1004(P04R2)2. 

The  result  is  an  ester  of  hexosediphosphoric  acid,  the  salts 
of  which  have  been  more  closely  investigated  by  Young 
(Biochem.  Z.,  1911,  32,  177). 

During  the  fermentation,  the  hexosediphosphate  accumulates 
in  the  solution,  but  as  soon  as  fermentation  ceases,  this  ester 
undergoes  hydrolysis  in  the  yeast  juice,  thus. 

(2)          C6Hi004(P04R2)2+2H20  =  C6Hi206+2R2HP04. 

This  hydrolysis  is  effected  by  a      hexosephosphatase."     These 
reactions  will  be  considered  further  in  Chapter  VII. 

Occurrence.  Zymases  do  not  only  occur  in  yeasts,  but  are 
extraordinarily  widespread  throughout  the  whole  of  the  animal  and 
vegetable  kingdoms.  There  is  now  scarcely  room  for  doubt  that  the 
combustion  of  sugar  in  the  animal  organism  and  also  in  higher  plants 
begins  with  decompositions  completely  analogous  to  those  brought  about 
by  yeast.  Deviation  from  these  occurs  only  in  the  final  phase  of  the 
reaction,  since  in  living  animal  organs  and  living  plants  alcohol  is  formed 
only  when  oxygen  is  lacking.  On  the  other  hand,  the  intramolecular 
respiration  of  sugar  is  absolutely  identical  with  alcoholic  fermentation. 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  55 

This  v.ew,  as  far  as  the  higher  plants  are  concerned,  is  due  especially  to 
E.  Godlewski,  Palladin  and  Kostytschew.  Also  the 
change  known  as  glycolysis,  occurring  in  the  animal  body,  must  be 
closely  allied  to  fermentation. 

Animal  glycolytic  enzymes  or  zymases  have  been  prepared  especially 
from  the  blood,  spleen,  pancreatic  tissues  and  muscle. 

Mme.  N.  Sieber  (H.,  1903,  39,  484;  1905,  44,  500)  has 
obtained  three  glycolytic  enzymes  in  the  form  of  stable  powders 
from  blood-fibrin  and  spleen: 

(a)  soluble  in  water, 

(b)  soluble  in  neutral  salt  solutions,  and 

(c)  soluble  in  water  or  alcohol  (peroxydase) . 

All  these  enzymes  contain  nitrogen  and  give  the  reactions 
of  the  proteins.  Their  ash  contains  iron,  manganese  and  phos- 
phoric acid,  but  not  copper.  The  second  of  them  gave  the  fol- 
lowing mean  composition:  C,  52%;  H,  7-5%  and  N,  15%. 

Tincture  of  guaiacum  is  turned  blue  directly  by  enzymes 
(a)  and  (b),  but  only  in  presence  of  hydrogen  peroxide  by  (c). 
11  6  h  m  a  n  n  and  S  p  i  t  z  e  r  '  s  reagent  (a  dilute  alkaline 
solution  of  a-naphthol  and  paraphenylenediamine)  is  coloured 
by  all  three  enzymes  in  absence  of  hydrogen  peroxide.  It  is 
doubtful  if  these  oxydase-  or  peroxydase-reactions  are  related 
in  any  way  to  the  ability  to  bring  about  the  combustion  of 
sugar.  In  any  case  the  essential  constituents  of  the  enzyme- 
complexes  obtained  by  Mme.  Sieber  are  fermentati^on- 
enzymes  and  not  oxidation  enzymes. 

Further  the  glycolytic  enzyme  found  by  Cohnheim 
(H.,  1903,  39,  336;  1904,  42,  401)  in  muscle,  from  which  he 
extracted  it  in  an  inactive  state,  is  not  oxydasic  in  character. 
It  is  a  decided  endo-enzyme  like  yeast-zymase  and  is  obtained 
from  the  frozen,  subdivided  muscle  by  pressing  or  by  extraction 
with  an  ice-cold,  isotonic  solution  of  sodium  oxalate;  the  oxalic 
acid  must  then  be  precipitated  with  the  calculated  quantity 
of  calcium  chloride  (H.,  1906,  47,  253).  Corresponding  with 
this  enzyme,  there  exists  a  co-enzyme  or  activator  which,  like 
that  of  yeast-zymase,  is  thermostable.  Cohnheim  pre- 
pared it  from  the  pancreatic  tissue  of  the  cat. 

The  objections  raised  by  Glaus  and  E  m  b  d  e  n  (Hofm, 
Beitr.,  1906,  6,  214  and  343)  to  the  investigations  of  Cohn- 
heim have  been  refuted  by  the  latter  author.  Reference 


56  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

must  be  made  to  Stoklasa's  work  (Chem.  Ber.,  1905,38, 
664;  Arch.  f.  Hygiene,  1904,  50,  165)  which  likewise  indicates 
the  existence  of  a  zymase  in  the  muscles  and  also  in  the  milk. 

It  is,  however,  remarkable  that  such  a  capable  experimenter 
as  A.  Harden,  working  in  conjunction  with  H  .  Maclean 
(Journ.  of  Physiol.,  1911,  42,  64),  was  unable  to  detect  alcoholic 
fermentation  by  animal  tissues  (liver,  kidneys,  pancreas,  flesh, 
etc.)  or  by  juices  or  powders  prepared  from  them. 

That  the  intramolecular  respiration  of  plants  is  to  be  regarded 
as  a  zymase  fermentation  has  already  been  mentioned  (Pal- 
ladin  and  Kostytschew,  H.,  1906,  48,  214;  Kost- 
ytschew,  Bot.  Ber.,  1908,  26,  167).  Also  by  means  of 
seedlings  of  Hordeum  distichum,  Pisum  sativum 
and  Lupinus  luteus,  Stoklasa,  Ernest  and  C  h  o  - 
c  en  sky  (H.,  1907,  50,  303;  1907,  51,  156)  have  clearly  shown 
the  action  of  the  enzymes  of  intramolecular  respiration.  The 
arguments  opposed  to  Stoklasa's  earlier  experiments  can 
scarcely  be  advanced  against  the  work  just  referred  to. 

Pal  lad  in  (Bot.  Ber.,  1905,  23,  240)  holds  the  view  that 
the  carbon  dioxide  respired  by  plants  arises  in  three  ways:  1. 
By  the  enzymes  combined  with  the  protoplasm;  Palladin 
calls  this  portion,  nucleo-carbon  dioxide  and  the  corresponding 
enzymes,  "  carbonases."  2.  By  the  protoplasm  itself  (apparently 
directly)  under  the  influence  of  various  irritants — irritant-carbon 
dioxide.  3.  By  the  action  of  oxydases.  In  another  place 
(Zeitschr.  f.  physikal.  Chem.,  1909,  69,  187,  Arrhenius- 
Festschrift),  the  author  has  indicated  that  he  is  unable  to  agree 
entirely  with  P  a  1 1  a  d  i  n  '  s  views,  especially  as  regards  car- 
bonase  and  the  role  of  oxydases  and  reductases  in  the  respiration 
process.  The  opportunity  must,  however,  not  be  neglected  to 
direct  attention  to  the  many  remarkable  observations  com- 
municated by  Palladin  to  the  Berichte  der  deut.  botan. 
Gesellschaft. 

Preparation  of  yeast-juice.  One  kilo  of 
washed  and  well-pressed  bottom-yeast  is  mixed  with  1  kilo 
of  fine  sand  and  300  grms.  of  kieselguhr  and  is  ground,  in  4-6 
lots,  in  a  large  mortar  until  the  mass  becomes  soft  and  doughy. 
In  this  way  a  large  proportion  of  the  yeast-cells  are  broken. 
The  dough  is  then  enveloped  in  a  press-cloth  and  pressed  in 
a  hydraulic  press,  the  pressure  being  raised  to  about  90  kilos 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  57 

per  sq.cm.  and  maintained  at  this  value  for  an  hour.  About 
400  c.c.  of  a  clear,  pale-brown,  viscous  juice,  containing  only  a 
very  small  number  of  living  cells,  are  thus  obtained  (  E  d  u  a  r  d 
and  Hans  Buchner  and  M  .  H  a  h  n ,  Die  Zymase- 
garung,  Munich,  1903,  p.  58). 

From  the  yeast-juice  the  zymase  can  then  be  precipitated 
with  alcohol  or  acetone,  which,  however,  throws  down  a  large 
amount  of  proteins,  carbohydrates  and  salts  at  the  same  time. 
In  order  to  obtain  very  active  preparations,  the  yeast-juice  must 
be  poured  into  a  large  excess  of  acetone  (10  volumes) ; .  only  in 
this  way  can  the  precipitate  formed  be  obtained  so  free  from 
water  that  the  chemical  changes  occurring  in  it  are  reduced 
to  a  minimum  (Buchner  and  Duchacek,  Biochem.  Z., 
1908,  15,  221). 

The  following  simple  method  for  obtaining  zymase-prepara- 
tions  has  been  recently  discovered  by  von  Lebedew  (C. 
R.,  1911,  152,  49;  Bull.  Soc.  Chim.,  1911,  [iv],  9,  744): 

It  consists  in  drying  the  yeast  at  a  temperature  of  25-30°  and 
macerating  with  water  for  2  hours  at  35°;  the  filtered  liquid 
exhibits  considerable  fermentative  activity.  According  to  the 
author's  experience,  Munich  yeast  (from  Schroder's  factory) 
and  many  other  yeasts  are  suitable  for  this  purpose,  but  this  is 
not  the  case  with  all  yeasts. 

According  to  Rinckleben  (Chem.  Zeitung,  1911,  35, 
1149),  zymase  can  also  be  obtained  by  plasmolysing  fresh  yeast 
with  glycerol. 

Preparation  of  Permanent  Yeast.  In  addi- 
tion to  those  described  above,  another  method  is  known  by  which 
the  fermentative  activity  of  yeast-cells  can  be  separated  from 
the  true  life  functions.  When  yeast  is  introduced  into  alcohol 
or  acetone  (Albert,  Chem.  Ber.,  1900,  33,  3775),  the  cells 
are  killed  without  their  fermenting  power  being  destroyed.  Well 
pressed  yeast  (500  grms.)  is  thoroughly  disintegrated,  placed 
on  a  hair-sieve  and  the  whole  dipped  into  a  basin  containing 
3  litres  of  acetone.  By  alternately  raising  and  lowering  the 
sieve  in  the  liquid,  and  by  means  of  a  small  brush,  the  whole  of 
the  yeast-cells  are  passed  through  the  sieve  in  3  or  4  minutes. 
The  yeast  is  then  left  in  the  acetone  for  10  minutes,  being  fre- 
quently stirred  meanwhile.  Most  of  the  liquid  is  next  poured 
off  and  the  yeast,  after  being  pumped  as  dry  as  possible,  is  again 


58  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

treated  with  acetone,  filtered  and  drained.  The  residue  is  well 
kneaded  with  250  c.c.  of  ether,  filtered,  dried  in  the  air,  finely 
ground  and  then  dried  for  24  hours  at  45°. 

This  preparation  has  been  placed  on  the  market  under  the  name 
of  "  zymin"  by  A.  Schroder,  of  Munich. 

The  best  antiseptics  to  use  with  yeast-juice  are  toluene  and 
thymol. 

Lactic    Acid   B  ac  ter  ia-zymase 

This  is  the  enzyme  by  means  of  which  lactic  acid  bacteria 
are  enabled  to  decompose  sugar  into  lactic  acid.  The  enzymic 
nature  of  this  transformation  was  demonstrated  by  Buchner 
and  Meisenheimer  (Chem.  Ber.,  1903,  36,  635;  Lieb. 
Ann.,  1906,  349,  125);  they  succeeded  in  obtaining  an  active 
permanent  preparation  of  Bacillus  Delbriickii  which, 
however,  only  gave  rise  to  small  quantities  of  ^-lactic  acid. 

Preparation.  The  organism  was  cultivated  at  40-45° 
in  wort  prepared  from  malt  and  rye. 

The  bacteria  were  subsequently  separated  by  means  of  a 
centrifuge  and  dried  on  a  porous  tile.  The  mass  was  then  intro- 
duced into  15-20  times  its  weight  of  acetone,  with  which  it 
was  ground  for  10-15  minutes,  the  bacteria  being  pumped  dry, 
washed  with  ether  and  dried  in  a  vacuum. 


OXYDASES 

The  action  of  oxydases  is  assumed  in  changes  of  very  dif- 
ferent kinds:  oxidations  of  purine  bases,  conversion  of  alcohols 
and  aldehydes  into  acids,  transformation  of  simple  and  substituted 
phenols,  amines,  amino-acids,  and  derivatives  of  these  com- 
pounds into  quinone  derivatives. 

The  following  kinds  are  thus  to  be  distinguished: 

1.  Purine-oxydases. 

2.  Alcoholases. 

3.  Aldehydases. 

4.  Phenolases. 

5.  Tyrosinase. 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  59 

To  these  true  oxydases  must  be  added  the  peroxydases, 
which  can  hardly  be  separated  sharply  from  the  oxydases,  and, 
especially  with  the  latter,  there  remains  still  a  good  deal  that 
is  not  clear,  so  that  the  division  of  this  section  can  be  considered 
only  as  a  provisional  one. 

For  the  recognition  of  oxydases  use  has  been  made  of  a 
number  of  reactions,  some  of  which  are  referred  to  below;  they 
are  not,  however,  given  by  all  oxydases,  which  are,  to  a 
great  extent,  specific  in  their  action. 

Blue  coloration  of  guaiacum  tincture  or  of  a-guaiaconic  acid;  violet 
coloration  of  tetramethylparaphenylenediamine;  brown  coloration  of 
m-  and  p-phenylenediamine  alone  or  in  presence  of  hydrogen  peroxide 
(A  so);  reddening  of  aniline  acetate  (C.  R.,  1896,  123,  315)  and  blue 
coloration  of  a-naphthol  (B  o  u  r  q  u  e  1  o  t ,  C.  R.,  1896, 123,  423;  Soc. 
BioL,  1896,  46,  896);  reddening  of  alo'in  (S  c  h  a  e  r  ,  Arch,  der  Pharm., 
1900,  38,  42;  K  a  s  1 1  e);  oxidation  of  phenolphthalin  to  phenolphthalein 
(K  a  s  1 1  e  and  S  h  e  d  d  ,  Amer.  Chem.  Journ.,  1901,  26,  526) ;  oxida- 
tion of  benzidine ;  leucorosolic  acid  (K  a  s  1 1  e,  Public  Health  and  Marine 
Hospital  Service  of  the  U.  S.  Hygienic  Lab.  Bull.,  1906,  No.  26,  7-22), 
hydroquinone,  pyrogallol,  and  guaiacol;  oxidation  of  leuco-malachite 
green  to  malachite  green  (quantitative,  spectro-photometric  method  of 
von  Czyhlarz  and  von  Fii  r  t  h  ,  Hofm.  Beitr.,  1907,  10,  358); 
oxidation  of  aldehydes,  e.g.,  salicylic  aldehyde  (S  c  h  m  i  e  d  e  b'e  r  g  , 
Arch.  f.  exp.  Path.,  1881,  14,  288,  379),  formaldehyde  (Pohl;  Arch.  f. 
exp.  Path.,  1896,  38,  65);  oxidation  of  arsenious  to  arsenic  acid;  forma- 
tion of  diaminophenacin  from  o-phenylenediamine,  and  of  indophenol 
from  p-phenylenediamine  and  a-naphthol  (R  6  h  m  a  n  n  and 
S  p  i  t  z  e  r ,  Chem.  Ber.,  1895,  28,  567). 

The  separation  of  iodine  from  potassium  iodide  has  also  been  regarded 
as  an  oxydase  reaction.  But  this  action,  as  has  been  pointed  out  by 
A  s  o  and  more  recently  by  Wolff  and  d  e  S  t  o  e  c  k  1  i  n  ,  C.  R., 
1908,  146,  1415),  must  be  attributed  to  the  nitrous  acid  occurring  in 
plant  juices. 

As  regards  the  oxidising  enzymes  of  the  purine 
bases,  the  action  of  xanthine  -  oxydase  is  best  under- 
stood, owing  especially  to  the  work  of  B  u  r  i  a  n  .  This  enzyme 
does  not  attack  uric  acid,  which  is  so  readily  broken  down  by 
ordinary  oxidising  agents.  B  u  r  i  a  n  supposes  it  to  be  quite  a 
general  oxidising  enzyme  for  (cyclic?)  amidines  which  is  unable 
to  attack  the  double  linking  between  the  (4)  and  (5)  carbon 
atoms  of  the  purine  nucleus: 


60  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

NH— CO  NH— CO  NH— CO 

CH    C  (4)— NH\               CO     C— NHv  CO     C— NH\ 

||        ||                >CH          |         ||          >CH         |         ||  >CO 

N C  (5) W  NH— C W  NH— C— NEK 

Hypoxanthine  Xanthine  Uric  acid 

An  enzyme  which  oxidises  uric  acid  to 
allantoin  was  obtained  by  Wiechowski  and  Wiener 
(Hofm.  Beitr.,  1907,  9,  232,  247,  295)  from  animal  organs  by 
treating  the  powdered  organ  (the  cells  being  completely  ruptured) 
with  0  •  05%  soda  solution. 

The  nitrates  obtained  after  grinding  with  the  soda  solution 
are  inactive,  but  if  the  emulsions  are  subjected  to  dialysis  into 
0-05%  soda  solution  for  5-6  days,  the  enzyme  of  dog's  liver 
passes  completely,  and  that  of  ox-kidneys  partially,  into  the 
filtrate.  Enzymic  liquids  were  also  obtained  by  centrifugating 
the  emulsions.  Repeated  precipitation  and  nitration  yielded  a 
protein-free  preparation,  which  exhibited  the  total  enzymic 
activity  of  the  starting  material. 

Alcoholase  :    Oxydase  of  Acetic  Bacteria. 

This  enzyme  oxidises  ethyl  alcohol  to  acetic  acid  and  thus 
catalyses  a  reaction  'for  which  energetic  oxidising  agents  are 
otherwise  necessary.  The  enzyme  has  not  yet  been  separated 
from  the  bacteria,  but  its  existence  can  be  proved  by  killing 
the  bacteria  with  acetone  (Buchner  and  M  e  i  s  e  n  - 
h  e  i  m  e  r  ,  Chem.  Ber.,  1903,  36,  637;  B  u  c  h  n  e  r  and  Gaunt, 
Lieb.  Ann.,  1906,  349,  140). 

Preparation.  Large  quantities  of  acetic  bacteria  (best 
Bacterium  aceti,  which  can  always  be  obtained  by 
leaving  beer  in  a  glass  dish  exposed  to  the  air)  are  cultivated 
on  beer-wort  to  which  4%  of  alcohol  and  1%  of  acetic  acid 
have  been  added.  As  culture-vessels,  shallow  glass  basins,  as 
wide  as  possible,  are  most  suitable;  these  are  covered  with  a 
layer  of  cotton  wool  in  order  to  prevent  the  entry  of  germs 
from  the  air.  In  the  course  of  4  or  5  days  after  inocu- 
lation, the  bacteria  form  a  fairly  thick  coating  on  the  surface  of 
the  nutrient  solution.  The  clear  liquid  underneath  is  syphoned 
off  and  the  residue  centrifuged  to  remove  most  of  the  liquid; 
the  brittle  bacterial  membranes  are  then  washed  superficially 
with  water  and  dried  on  porous  tile.  On  the  following  day  the 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  61 

mass  is  introduced  into  10-20  times  its  weight  of  acetone,  rubbed 
to  a  fine  powder  and  left  for  10  minutes,  after  which  it  is  filtered, 
washed  with  ether  and  dried  in  a  vacuum  over  sulphuric  acid. 
This  procedure  yields  a  yellowish  powder  which,  however,  only 
oxidises  very  small  quantities  of  ethyl  alcohol. 

Aldehydases.  Aldehydes,  e.g.,  benzaldehyde  and  sal- 
icylic aldehyde,  are  oxidised  by  the  extracts  of  many  animal 
organs,  but  it  is  doubtful  if  this  is  an  enzyme  action.  See  the 
remarks  of  B  a  c  h  (Chem.  Ber.,  1904,  37,  3791)  arid  the  results 
of  Dony-Henault  and  van  Duuren  (Bull.  Acad. 
roy.  Belgique,  1907,  577). 

Of  the  vegetable  oxydases  mention  must  first  be  made  of 
the  laccase  from  the  lac-tree,  which  was  discovered  by  Y  o  s  h  i  d  a 
and  studied  in  detail  by  Bertrand,  who  gives  the  follow- 
ing method  for  its  preparation  (Ann.  de  Chim.  et  de  Phys., 
1897,  [vii],  12,  115). 

The  thick  sap  of  Rhus  succedanea  is  mixed  with 
5-6  times  its  volume  of  alcohol,  by  which  means  the  laccase 
is  precipitated,  whilst  the  phenolic  derivatives  which  produce 
the  blackening  of  the  lac  pass  into  the  alcoholic  solution.  The 
solution  is  filtered  through  a  cloth  and  the  residue  washed  on 
the  cloth  several  times  with  alcohol.  It  is  then  taken  up  in 
cold  distilled  water,  only  a  small  quantity  of  black  substance 
remaining  undissolved.  The  filtered  solution  is  again  precipitated 
with  alcohol  in  large  excess,  the  precipitate  being  collected  on 
the  filter  and  dried  in  a  vacuum. 

Laccase  is  obtained  as  a  white  substance  having  a  neutral 
reaction  and  readily  soluble  in  water.  Bertrand  regards  it 
as  a  protein,  although  his  preparation  contained  only  0-44% 
of  nitrogen.  He  assumes  its  composition  to  be  as  follows : 

Moisture  (determined  at  120°) 7-40% 

Gum  (arabans  and  galactans) 84-95 

Laccase 2-50 

Ash 5-17 

The  most  essential  constituent  of  the  ash  is  manganese, 
which  is  present  to  the  extent  of  2-5%. 

Laccase  is  extremely  sensitive  to  acids  (cf.  Chapter  III)  and 
is  destroyed  by  short  boiling  of  its  solution. 


62  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

From  other  plants,  such  as  Medicago  sativa  and 
Lolium  perenne,  Bertrand  obtained  preparations 
which  he  termed  laccases,  since,  in  neutral  solution  and  in 
presence  of  manganese  salts,  they  accelerate  the  oxidation  of 
polyphenols.  As  the  author  has  pointed  out  (H.,  1909, 
61,  1),  they  are  quite  different  from  R  h  u  s  -  1-acease. 

Preparation  of  Medieago-lacca.se.  The 
fresh  plants,  at  the  commencement  of  the  flowering  stage,  are 
chopped  up  and  pressed.  On  standing,  the  juice  obtained 
deposits  dark  flocks,  from  which  it  is  separated  by  filtration. 
Alcohol  is  then  added,  the  abundant  precipitate  thus  formed 
being,  to  a  large  extent,  taken  up  in  water  and  again  precipi- 
tated. This  procedure  is  repeated  thrice,  the  preparation  ob- 
tained, after  drying  in  a  desiccator,  being  a  white,  dusty,  highly 
hygroscopic  powder,  soluble  in  water  with  great  readiness. 
According  to  E  u  1  e  r  and  B  o  1  i  n  ,  it  consists  mainly  of  the 
calcium  salts  of  aliphatic  hydroxy-acids.  The  separation  of  the 
mixture,  effected  by  fractional  crystallisation  of  the  correspond- 
ing barium  salts,  shows  it  to  contain  glycollic,  citric,  malic,  and 
mesoxalic  acids.  The  oxidising  action  of  these  salts  is  described 
in  Chapter  IV. 

Numerous  attempts  have  been  made  to  prepare  laccases  artificially. 
According  to  Bertrand,  the  oxidising  agent  of  many  plants  is 
composed  of  manganese  and  a  protein  with  a  specific  action.  T  r  i  1 1  a  t 
(C.  R.,  1904,  138,  94,  274)  assumed  that  the  laccase  regarded  as  a 
specific  enzyme  can  be  replaced  by  any  protein  or,  at  any  rate,  by 
certain  classes  of  proteins;  he  has,  however,  no  good  foundation  for  this 
view. 

Especially  on  the  ground  of  his  own  experiments  on  R  h  u  s  -  laccase, 
the  author  also  regards  as  unsuccessful  Dony-He*nault's  attempts 
to  attribute  the  action  of  laccase  to  the  alkalinity  of  the  preparations 
and  thus  to  show  that  lac  case-action  is  only  an  oxidation  by  means  of 
manganese  and  hydroxyl-ions.  The  laccase  preparations  obtained  by 
Bertrand's  method  are  not  alkaline,  but  are  extremely  active. 

Further  efforts  to  prepare  artificial  oxydases  and  peroxydases  have 
been  made  by  M  a  r  t  i  n  a  n  d  (C.  R.,  1909,  148,  182),  Wo  1  f  f  (C.  R., 
1908,  147,  745),  and  d  e  S  t  o  e  c  k  1  i  n  (C.  R.,  1908,  147,  1489). 

With  other  oxydases,  so  little  has  been  done  as  regards  puri- 
fication and  isolation  that  is  has  not  been  decided  to  what  class 
of  bodies  these  substances  belong 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  63 

Slowtzoff  (H.,  1900,  31,  227)  attempted  to  prepare  the  "  lac- 
case"  of  potatoes  in  a  pure  state,  his  purest  preparation  containing 
12- 8%  NandO- 53%  S. 

Sarthou  (J.  de  Pharm.  et  Chim.,  1900,  [vi],  11,  482,  583;  1901, 
[vi],  13,  464)  obtained  quite  different  numbers  for  his  schinoxydase, 
namely,  6-28%  N,  0-2%  S,  and  1-34%  ash,  from  which  he  concluded 
the  enzyme  to  be  a  nucleoprotein.  A  s  o  and  L  o  e  w  regard  the 
oxydases  as  albumoses.  But  Rosenfeld's  investigations  (Disser- 
tation, St.  Petersburg,  1906)  on  the  oxydase  of  the  radish  (Raphanus 
s  a  t  i  v  u  s)  appear  to  indicate  that  this  enzyme  does  not  belong  to 
the  proteins.  According  to  R  o  s  e  n  f  e  1  d  the  oxydase  would  be  a 
crystalline  substance,  containing  C,  N,  S,  P,  and  K  but  not  Fe  or  Mg, 

S  pn  t  z  e  r  came  to  the  conclusion  that  the  oxydase  of  the  liver 
is  a  nucleoprotein,  but  this  enzyme,  which  effects  the  oxidation  of 
salicylic  aldehyde  to  salicylic  acid,  was  further  purified  by  J  a  c  o  b  y 
(H.,  1900,  30,  135).  It  is  found  to  be  soluble  in  water  and  non-diffusible, 
and  to  become  inactive  on  heating,  while  it  does  not  give  the  reactions 
characteristic  of  the  proteins. 

According  to  Tschirch  and  Stevens  (Arch,  der  Pharm.,  1905, 
43,  504),  the  oxydase  of  Japanese  lac  shows  the  pyrrole  reaction. 
Bach  and  C  h  o  d  a  t  are  of  opinion  that  the  oxydase  consists  of  a 
peroxydase  and  an  oxygenase,  the  former  alone  giving  the  pyrrole 
reaction  (Bach,  Chem.  Ber.,  1908,  41,  226). 

Theoenoxydaseof  apples  (L  i  n  d  e  t ,  C.  R.,  1895,  120,  370), 
pears,  plums,  grapes,  and  the  fungus  Botrytis  cinerea,  parasitic 
to  grapes,  must  be  a  tannin-oxydase ;  by  its  action  the  flesh  of  the 
fruit  is  turned  brown  on  exposure  to  the  air. 

Oxydases  of  doubtful  individuality  have  been  detected  in 
numerous  plants,  e.g.,  Arum  maculatum,  olives  ("  olease  ")> 
barley  and  malt  ("  spermase  "),  coffee  beans  and  yeast. 

Should  B  a  1 1  e  1 1  i '  s  observation  (C.  R.,  1904,  138,  651)  on 
the  oxidation  of  formic  acid  to  carbonic  acid  by  oxydases  in 
presence  of  hydrogen  peroxide  be  confirmed,  an  interesting 
organic  oxidation  will  present  itself.  In  this  connection,  mention 
may  be  made  of  Loevenhart's  discovery  (Chem.  Ber., 
1906,  39,  130)  that  formic  acid  is  oxidised  to  carbon  dioxide 
by  hydrogen  peroxide  in  presence  of  iron,  copper,  etc. 

A  very  remarkable  oxidation,  which  is  not  yet  understood, 
has  been  described  by  Z  a  1  e  s  k  i  and  R  e  i  n  h  a  r  d  (Bio- 
chem.  Z.,  1911,  33,  449);  it  consists  in  the  oxidation  of  oxalic 
acid  in  1%  solution  to  carbonic  acid. 


64  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

For  information  concerning  other  oxydases,  see  Battelli 
and  Stern's  resume  in  Ergeb.  der  Physiol.,  1912,  12,  95. 

Ty  rosinase 

(G.Bertrand,  Bull.  Soc.  Chim.,  1896,  [iii],  15,  791.) 
From  tyrosine  and  its  derivatives  this  enzyme  forms,  by  oxidation, 
melanins — dark-coloured  substances  of  unknown  chemical  com- 
position. 

Interesting  data  concerning  the  chemical  aspect  of  the  action 
of  tyrosinase  have  recently  been  obtained  by  Abderhalden 
and  Guggenheim  (H.,  1908,  54,  331).  They  were  able 
to  show  that  ozone  and  tyrosine  alone  do  not  yield  melanins, 
but  that  these  are  synthesised  by  oxidation  from  tyrosine  or 
polypeptides  containing  it,  on  the  one  hand,  and  from  phenols 
or  amino-acids  on  the  other.  Similar  colorations  are  obtained 
if  the  oxidation  is  effected  with  potassium  dichromate  instead 
of  tyrosinase,  and  it  is  hence  probable  that  tyrosinase  contains 
an  oxidising  agent,  the  action  of  which  is  exerted  along  with 
that  of  the  amino-acids  of  the  plant-juice. 

Tyrosinase  attacks  not  only  Z-tyrosine  itself,  but  also  dl- 
tyrosine,  tyrosine  anhydride  (Bertrand  and  Rosen- 
blatt; Chodat),  and  a  large  number  of  polypeptides 
containing  tyrosine.  Suprarenin  (adrenaline)  is  likewise  oxidised, 
and  all  the  cresols,  resorcinol,  m-toluidine,  o-,  m-  and  p-xylenols, 
thymol,  carvacrol  and  naphthol;  also,  according  to  Ber- 
trand, phenol. 

The  opinion  expressed  by  Gonnermann  that  the 
specific  action  of  tyrosinase  consists  of  a  hydrolysis,  which  is 
then  followed  by  an  oxidation  (not  specific),  has  been  combated 
by  Chodat  and  Bach. 

According  to  Chodat,  Zahorski  and  F  r  e  e  d  e  - 
rickz  (Arch.  Sci.  phys.  nat.,  1909,  27,  306),  the  specificity  of 
tyrosinase  is  conditioned  by  the  presence  of  an  activator  which 
is  stable  to  heat. 

For  the  preparation  of  the  enzyme,  the  disintegrated 
fungus  is  extracted  with  water  and  the  extract  precipitated 
with  alcohol. 

Occurrence.  In  numerous  fungi  of  the  species  Boletus, 
Russula,  Lactarius,  Coprinus,  Paxillus,  etc.  Also 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  65 

in  Merulius  lacrimans,  beet-juice,  dahlia  bulbs,  potato 
skins,  and  V  i  c  i  a  f  a  b  a  .  Tyrosinase  is  often  accompanied  by  lac- 
cases. 

From  sepia,  C.  Neuberg  has  extracted  an  enzyme 
which  resembles  tyrosinase  and  acts  on  adrenaline. 

PEROXYDASES 

Under  this  name  are  included  those  substances  which  activate 
peroxides.  Their  typical  reaction  is  the  transference  of  oxygen 
from  hydrogen  peroxide  to  guaiaconic  acid  or  to  polyphenols. 

Bach  and  Chodat  (Chem.  Ber.,  1903,  36,  600)  have 
given  a  method  for  the  preparation  of  peroxydase  from  pump- 
kins and  horse-radish  roots.  Bach  and  Tscherniak 
have  recently  (Chem.  Ber.,  1908,  41,  2345)  obtained  a  peroxy- 
dase in  the  following  manner: 

Thirty  kilos  of  turnips  were  pounded  up  and  pressed  and 
the  juice  obtained  (20  litres)  mixed  with  2  litres  of  96%  alcohol 
in  order  to  coagulate  the  gummy  matters.  After  nitration, 
the  alcoholic  juice  was  precipitated  by  130  litres  of  strong 
alcohol,  the  precipitate  being  filtered  off,  washed  with  alcohol 
and  freed  from  precipitant  in  a  vacuum.  The  crude  peroxydase 
thus  obtained  (52  grms.)  was  kneaded  with  600  c.c.  of  water, 
only  a  small  portion  of  the  substance  passing  into  solution. 
The  undissolved  residue  was  filtered  off  and  washed  with  a  little 
water,  and  to  600  c.c.  of  the  filtrate,  containing  only  about  7 
grms.  of  dry  matter,  40  grms.  of  powdered  basic  lead  acetate 
were  added;  the  precipitate  was  pumped  off  and  the  clear  filtrate 
(600  c.c.)  treated  with  powdered  sodium  carbonate  (21  grms.) 
until  no  further  turbidity  was  produced.  The  alkaline  filtrate 
was  dialysed  through  parchment  into  distilled  water.  After 
13  days  the  dialysate  (670  c.c.)  was  mixed  with  4-5  litres  of 
99%  alcohol,  and  the  precipitate  thus  formed  collected,  after 
24  hours,  on  a  hardened  filter,  washed  with  absolute  alcohol 
and  freed  from  the  latter  in  a  vacuum. 

This  preparation  contained  7  •  87%  of  water,  81  •  66%  of  organic 
matter  and  1-47%  of  ash;  the  percentage  of  nitrogen,  calculated 
on  ash-free  material,  was  3  •  44. 

The  peroxydase  prepared  by  E.  de  Stoecklinin 
C  h  o  d  a  t '  s  laboratory  from  Cochlearia  armoracia 


66  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

contained  1 1  •  41%  of  water,  65  •  88%  of  organic  matter  and  22  •  71% 
of  ash;  the  nitrogen-content  was  3-43%.  The  activating  power 
of  this  peroxydase  was  only  one-tenth  part  of  that  of  the  above- 
mentioned  preparation  of  Bach.  Neither  de  Stoecklin 
nor  C  hod  at  (Schweiz.  Woch.  Chem.  u.  Pharm.,  1905,  43) 
obtained  protein  reactions  with  their  peroxydases. 

The  author's  experience  (H.,  1909,  61,  1)  indicates 
that  dialysis  is  the  best  known  method  of  purifying  peroxydase 
preparations.  From  horse-radish  E  u  1  e  r  and  B  o  1  i  n  ob- 
tained a  preparation  which  increased  continuously  in  activity 
when  subjected  to  dialysis.  The  preparation  showing  the  greatest 
activity  per  unit  of  weight  contained  10-4%  N  and  2-5%  of  ash. 
Dialysis  is  rendered  far  more  effective  if  the  enzymic  juice  is 
previously  treated  with  kaolin  or  other  suitable  adsorption 
agent  in  order  to  remove  the  proteins.  D  e  1  e  a  n  o  (  Biochem. 
Z.,  1909,  19,  266)  proposes  the  use  of  colloidal  ferric  hydroxide 
for  this  purpose.  Bach  (Chem.  Ber.,  1910,  43,  362)  suggests 
the  preliminary  removal  of  the  gummy  matters  by  means  of 
magnesium  sulphate. 

During  recent  years  it  has  been  repeatedly  pointed  out  that 
the  action  of  peroxydases  can  be  obtained  partly  by  purely 
inorganic  materials  (see  Wolff,  C.  R.,  1908,  146,  781  and 
M  a  r  t  i  n  a  n  d  ,  C.  R.,  1909,  148,  182),  and  partly  by  synthetic 
organic  preparations  (de  Stoecklin,  C.  R.,  1908,  147, 
1489). 

As  Moitessier,  Lesser,  von  Fiirth  and  von 
C  z  y  h  1  a  r  z  ,  and  Bertrand  and  Rogozinski  (C. 
R.,  1911,  152,  148)  have  shown,  the  well-known  guaiacum-blue 
reaction  of  the  blood  depends  not  on  an  enzyme,  but  on  the 
haemoglobin;  it  appears  with  undiminished  intensity  after  boiling. 
Oxyhsemoglobin  acts  not  only  as  a  peroxydase  but  as  an  oxy- 
dase  as  well  (de  Stoecklin).  Further,  the  oxidation 
phenomena  in  milk  cannot  depend  on  the  presence  of  a  peroxydase. 

Thermolabile  peroxydases  do,  however,  exist  and  these  must 
for  the  present  be  classed  as  enzymes. 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  67 


CATALASES 

(  O  .  L  o  e  w  ,  Rep.  U.  S.  Dept.  of  Agric.,  1901,  No.  68.) 
After  the  ability  to  decompose  hydrogen  peroxide  had  been  long 
regarded  as  a  general  property  of  the  enzymes,  O  .  L  o  e  w 
demonstrated  the  existence  of  special  catalases.  The  discoverer 
distinguished  two  catalases  occurring  in  plants,  viz.,  an  a-catalase 
which  is  not  extractable  by  water  and  was  regarded  as  a  nucleo- 
protein,  and  a  g-catalase  soluble  in  water  which  was  regarded  as 
an  albumose. 

The  chemical  action  of  the  catalases  is  analogous  to  that  of 
the  colloidal  metals  (  B  r  e  d  i  g  ),  molecular  (inert)  oxygen  being 
formed,  together  with  water.  Ethyl  hydroperoxide  is  not  decom- 
posed (Bach  and  Chodat,  Chem.  Ber.,  1903,  36,  1757). 

Occurrence  :  Extremely  widespread  in  the  animal  and  vege- 
table kingdoms.  In  blood  (S  enter;  Lesser,  Zeitschr.  f.  Biol., 
1906,  48,  1) ;  in  numerous  organs  (K  a  s  1 1  e  and  Loevenhart  ; 
Liebermann;  Battelli,C.  R.,  1904,138,923);  in  milk(Raud- 
nitz,  Zeitschr.  f.  Biol.,  1901,  42,  91;  Reiss,  Zeitschr.  klin.  Med., 
1905,  56,  1;  Faitelowitz,  Dissertation,  Heidelberg,  1904). 
Catalase  is  also  found  in  virtually  all  plant-juices.  Especially  rich  in 
this  enzyme  are  many  leaves,  e.g.,  of  clover,  Rosa,  Picea,  which 
mainly  contain  the  insoluble  form  of  the  enzyme;  and  certain  seeds 
e.g.,  of  the  apple  and  peach,  in  which  the  enzyme  occurs  principally  in 
the  soluble  form.  Highly  active  catalases  are  obtained  from  fungi, 
e.g.,  Boletus  scaber  (E  u  1  e  r  ,  Arkiv  for  Kemi,  1904,  1,  357), 
and  from  the  lower  fungi,  yeasts,  and  bacteria. 

The  preparation  is  usually  carried  out  by  precipitating 
the  aqueous  extracts  with  alcohol.  S  e  n  t  e  r  gives  the  follow- 
ing method  of  obtaining  the  catalase  of  the  blood:  Defibrinated 
ox-blood  is  mixed  with  10  times  its  volume  of  carbonated  water 
and  left  over  night.  It  is  then  centrifuged  and  filtered  and  the 
liquid  precipitated  with  an  equal  volume  of  alcohol,  the  alcoholic 
solution  of  haemoglobin  being  poured  off  and  the  reddish-brown 
precipitate  repeatedly  washed  with  50%  alcohol.  The  precipitate 
is  dried  in  a  vacuum,  ground  to  a  fine  powder,  stirred  with 
water  and  allowed  to  stand  in  ice  for  2  or  3  days  in  order 
that  the  enzyme  may  be  extracted  completely.  The  solution 
is  filtered  through  hardened  filter-paper  until  it  becomes  quite 


68  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

clear;  the  faintly  yellow  liquid  thus  obtained  vigorously  decom- 
poses hydrogen  peroxide  with  evolution  of  oxygen. 

For  the  preparation  of  highly  purified  catalase-products, 
pig-  or  ox-fat  is  the  most  suitable  starting  material.  The  fat  is 
disintegrated  by  means  of  sand  and  extracted  with  water  at 
about  30°,  the  enzyme  being  then  precipitated  with  alcohol  and 
further  purified  by  repeatedly  dissolving  in  water  and  precip- 
itating with  alcohol  (Euler,  Arkiv  for  Kemi,  1904,  1,  357; 
Bach,  Chem.  Ber.,  1905,  38,  1878). 

In  his  first  experiments  ( 1  o  c  .  c  i  t . )  the  author  obtained 
a  preparation  which  still  showed  faint  protein  reactions  and 
contained  14-5%  N  and  1-2%  S  but  no  phosphorus.  A  prepa- 
ration made  from  defibrinated  blood  and  similarly  purified  gave 
14-1%  N.  A  continuation  (not  yet  published)  of  this  investi- 
gation has  yielded  enzyme-preparations  which  are  certainly  of 
a  higher  degree  of  purity.  The  nitrogen-content  diminishes  as 
the  purification  proceeds,  the  final  product,  which  exhibits  con- 
siderable activity,  containing  6  •  2%  N  and  exhibiting  M  i  1 1  o  n  's 
and  M  o  1  i  s  c  h  '  s  reactions.  Fat-catalase  appears  therefore 
to  be  not  a  protein,  but  mention  must  be  made  of  the 
opposite  results  obtained  by  Bach  (loc.  cit.).  The  question 
of  the  protein  character  of  catalase  hence  requires  further 
investigation. 

Reducin.g    Enzymes     (Reductases;    Reducase) 

A  substance  termed  "  philothion "  was  described  by  d  e 
Rey-Pailhade  and  was  regarded  by  him  and  also  by 
Pozzi-Escot  as  a  reducing  enzyme.  With  this  view, 
however,  the  author  is  unable  to  agree,  since  the  reactions  described 
by  de  Rey-Pailhade  and  Pozzi-Escot  are  not 
enzymic  in  character.  The  existence  of  reducing  enzymes  has, 
indeed,  not  yet  been  demonstrated  with  absolute  certainty. 
In  most  cases  in  which  reduction  has  been  observed,  no  attempt 
has  been  made  to  show  that  it  is  really  due  to  an  enzyme-action, 
i.  e.,  a  catalytic  reaction,  and  is  not  merely  a  stoichiometric 
reduction  by  a  readily  oxidisable  substance. 

A  substance  which,  at  70°,  accelerates  the  reduction  of  meth- 
ylene  blue  by  formaldehyde,  has  been  found  to  exist  in  milk 
(Schardinger).  That  this  is  a  catalytic  action  has  been 


SPECIAL  CHEMISTRY  OF  THE  ENZYMES  69 

rendered  probable  by  the  investigations  of  S.  Oppenheimer 
(Arb.  a.  d.  Inst.  f.  exp.  Therapie  in  Frankfurt,  1908,  4)  and 
of  Trommsdorff  (Centralbl.  f.  Bakt.,  1909,  49,  291). 
Schardinger's  reaction  is  also  hastened  by  colloidal 
platinum  or  iridium  (  B  r  e  d  i  g  and  S  o  m  m  e  r  ,  Zeitschr. 
f.  physikal.  Chem.,  1910,  70,  34). 

Bach  has  recently  (Biochem.  Z.,  1911,  31,  443)  endeavoured 
to  ascertain  if  Schardinger's  enzyme,  for  which  he 
proposes  the  name  perhydridase,  is  related  to  the  reduc- 
ing enzymes  of  the  liver  and  other  tissues.  He  is  of  the  opinion 
that  the  "  reducase  "  of  the  liver  is  a  mixture  of  enzymes  and 
contains  that  of  Schardinger.  Further,  the  same  reaction 
underlies  the  action  of  the  systems :  palladium — methylene  blue — 
hypophosphate — water;  palladium — methylene  blue — aldehyde 
— water;  milk-enzyme — methylene  blue — aldehyde — water,  this 
reaction  consisting  in  decomposition  of  the  water  by  the  oxidis- 
able  substance  with  the  help  of  a  catalyst  which  forms  a  labile, 
strongly  reducing  compound  with  the  hydrogen  of  the  water. 

In  the  researches  of  Abelous  and  his  collaborators  on 
horse-kidneys,  bacterial  action  was  not  excluded.  Indeed,  as 
Abelous  himself  stated  and  the  author  has  confirmed,  no 
reduction  takes  place  if  the  extract  is  filtered  through  a  Chamber- 
land  candle  (cf .  the  work  of  Heffter,  Arch.  f.  exp.  Path., 
Schmiedeberg-  Festschrift,  1908,  29). 

In  the  roots  of  plants  and  in  seedlings,  strongly  reducing 
substances  occur  but  these  are  not  enzymic  metabolic  products 
(O.  Schreiner  and  M.  Sullivan).  The  same  holds 
for  reduction  by  micro-organisms.  K  a  s  1 1  e  and  E  1  v  o  v  e 
(Amer.  Chem.  Journ.,  1904,  31,  606)  have  also  given  an  interesting 
study  on  the  reducing  actions  of  plant-juices. 

APPENDIX 

In  the  above  short  account,  mention  has  only  been  made 
of  those  enzymes,  of  the  individuality  and  mode  of  action  of 
which  something  definite  is  known.  A  large  number  of  enzymes, 
which  have  received  special  names,  have  not  been  considered, 
as  they  do  not  differ  essentially  from  the  better-known  repre- 
sentatives of  the  same  groups. 

But  certain  other  enzymes,  which  have  found  no  place  in  the 


70  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

main  enzyme  groups,  may  be  briefly  referred  to  here,  since  their 
further  study  appears  to  be  not  without  interest. 

1.  An   isomerising     enzyme,    which  converts  man- 
nose  into  glucose,  is  thought  by    Gat  in    (Soc.   BioL,   1908, 
65,  903),  to  exist  in  the  seeds  of    Borassus    flabelli- 
f  o  r  m  i  s  . 

2.  According  to   J.   Parnas    (Biochem.  Z.,  1910,  28,  274) 
the  liver  contains  a  soluble  enzyme  which  is  able  to  accelerate 
Cannizzaro's  aldehyde  transformation,  i.e.,  it  converts  aldehydes 
anaerobically  into  the  corresponding  alcohols  and  acids.     Par- 
nas    suggests     the     name     aldehydemutase,    whilst 
Battelli   and   Stern    (Biochem.  Z.,   1910,  29,   130)   apply 
the   term    aldehydase    to   a   similar   enzyme   studied   by 
them. 

3.  Kotake    (H.,   1908,  57,  378)  refers  to  an  enzyme  of 
ox-liver    which    demethylates    caffeine,    giving  xan- 
thine,  hypoxanthine,  ^-methylxanthine,  etc. 

4.  In  the  kidneys  and  liver,   Gottlieb    and  Stangas- 
singer   (H.,  1907,  52,  1;   1908,  55,  295,  322)  found  substances 
which   convert    creatine    into    creatinine    and   are 
apparently  enzymic  in  character. 


CHAPTER    II 
PHYSICAL    PROPERTIES    OF    THE    ENZYMES 

ALTHOUGH  the  courses  followed  by  most  enzymic  reactions 
can  be  represented  by  formulae  which  hold  for  catalyses  in  homo- 
geneous systems,  and  although  also  dynamics  as  yet  affords  little 
means  of  taking  the  state  of  solution  or  the  colloidal  condition 
of  the  enzymes  into  account,  yet,  in  experiments  with  enzymic 
liquids,  adsorption  phenomena  always  make  themselves  more 
or  less  strongly  felt  and  have,  indeed,  a  determining  influence 
on  the  general  chemical  behaviour  of  the  enzymes. 

Wherever  a  liquid  is  bounded  by  a  vaporous  space,  there 
is  formed  at  the  surface  a  layer  possessing  properties  different 
from  those  of  the  body  of  the  liquid —  this  layer  is  termed  the 
surface-layer.  The  latter  has  a  tendency  to  diminish,  and  it 
is  to  this  that  the  well-known  capillary  phenomena  are  due; 
on  the  interior  of  the  liquid  a  pressure  is  exerted,  termed  the 
internal  pressure. 

The  thickness  of  this  surface-layer,  which  differs  from  the 
remainder  of  the  liquid,  is  very  small. 

The  pressure  with  which  the  surface-layer  presses  on  the 
internal  liquid  is  also  very  small,  unless  the  relation  between 
the  surface  of  a  substance  and  its  volume — "  the  specific  sur- 
face " — is  very  large,  i.e.,  the  distribution  of  the  substance  is 
considerable.  The  latter  is  especially  the  case  with  the  so- 
called  colloidal  solutions. 

1  We  shall  begin  with  a  short  theoretical  consideration  of 
surface  phenomena,  employing  the  method  of  representation 
given  by  Maxwell. 

The  wire  EF  (Fig.  1)  is  to  be  regarded  as  capable  of  moving 
freely  along  the  rectangular  wire  A  BCD.  Within  the  frame 
EBCF  is  a  layer  of  liquid  which,  in  consequence  of  the  surface- 
tension,  tends  to  diminish  and  so  draw  the  side  EF  upwards. 

71 


72  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

The  weight  is  so  chosen  that  it  exactly  compensates  the  pull 
of  the  liquid  layer  on  the  wire;  increase  of  the  load  G  would 

then  result  in  rupture  of  the  layer  and 
decrease,  in  rise  of  EF. 

The  pull  of  the  layer  is  caused  by 
its  surface-tension,  and,  since  both 
surfaces  of  the  layer  are  active,  cor- 
responds with  double  the  surface- 
tension. 

If  now  the  movable  wire  is  displaced, 
by  means  of  the  weight  G,    from   its 
FIG  i  highest  position  BC  to  the  position  EF, 

an    amount    of  work    has   been   done 

against  the  surface-tension  which  is  proportional  to  G  (made 
equal  to  the  surface-tension)  and  to  the  magnitude  of  the  sur- 
face produced.1  This  work  evidently  represents  the  surface- 
energy  and  is  given  by : 

Surface-energy  =  Surface-tension  X  surface. 

The  surface-tension  may  also  be  expressed  by  the  energy 
acting  on  unit-surface  or  by  the  force  acting  on 
unit-length,  and  is  usually  given  in  dynes  per  cm. 

Owing  to  the  small  absolute  values  possessed  by  the  known 
surface-tensions  of  liquids  and  aqueous  solutions — about  100 

dvnes 

— the    surface-energy  of  a  liquid    attains   a   considerable 
cm. 

magnitude  only  when  the  surface  is  large,  and  hence  becomes 
comparable  with  the  other  forms  of  energy  of  a  substance  only 
when  the  ratio  of  surface  to  volume — the  "  specific  surface  " 
(Wo.  Ostwald ) — exceeds  a  certain  value,  namely,  about 
10,000. 

The  physiologically  most  important  and  most  interesting 
phenomena  in  this  region  are  met  with  not  in  pure  liquids,  but 
partly  in  solutions  and  partly  in  "  heterogeneous  systems,"  in 
which  a  substance  occurs  in  a  very  fine  state  of  division  and 
which  Wilh.  Ostwald  has  named  disperse  systems. 

Solutions.     It   can  be  stated  generally  that  the   con- 

1  The  surface-tension  of  liquids  is  independent  of  the  expanse  of  the 
surface. 


PHYSICAL  PROPERTIES  OF  THE  ENZYMES  73 

centration  at  the  surface  of  a  solution  is  different  from  that 
prevailing  inside  the  solution.  This  can  be  demonstrated  experi- 
mentally and  also  follows  from  thermodynamical  considerations. 
The  derivation  given  below  follows  that  given  by  M  i  1  1  n  e  r 
[Phil.  Mag.,  1907,  (6),  13,  96]  and  was  also  deduced  by  H  . 
Freundlich  in  his"  Kapillarchemie." 

Suppose  n  molecules  to  be  dissolved  in  a  certain  volume  v.    The 
concentration  has  then   not  the   uniform  value  --,  but  is  greater  (or 

smaller)  in  the  surface-layer  (or  interior).  If  the  excess  per  unit  of  surface 
w  is  indicated  by  a,  the  excess  of  concentration  in  the  whole  surface  will 

be  aw  and  the  concentration  in  the  interior  of  the  liquid,  c  =  -  . 

v 

Consideration  of  a  thermodynamic  cycle  leads  to  the  differential 
equation: 

dr        dp 


where  T  is  the  surface-tension  and  p  the  osmotic  pressure. 

This  expresses  the  fact  that  the  surface-tension  changes  with  the 
volume  and,  therefore,  also  with  the  concentration,  if  the  osmotic 
pressure  changes  with  the  magnitude  of  the  surface,  and  this  can  only 
happen  if  the  concentration  of  the  solution  depends  on  the  magnitude 
of  the  surface  and  is  therefore  different  from  the  concentration  in  the 
latter. 

If  equation  (I)  is  developed  as  a  function  of  c,  it  gives  rise  to 


dv    dc  dv         v   dc (II) 


dt    dr  dc         c 
dv    dc 
and 

-r~=T'7~- 'T" (HI) 

aw     dc  aw         v   dc 


The  excess  of  concentration  per  unit  of  surface  is  hence  given  by 

C?T         <j   dp 
dc         c   dc' 


(IV) 


If  the  change  cf  surface-tension  with  concentration  is  known,  a  can 
be  calculated  from  (IV).     If  the  osmotic  pressure  obeys  the  simple 

gas-laws,  then  -j-  =  RT,  and  hence 


—-      or      —  . 


74  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

So  far  as  the  surface-tension  of  inorganic  solutions  has  been  measured, 
it  exhibits  a  linear  increase  with  the  concentration,  i.e.,  —  is  positive 

and  constant  and  therefore  a  is  negative  and  —  constant.     Hence  we 
arrive  at  the  result: 

If  the  surface-tension  of  a  solution  dimin- 
ishes with  increasing  concentration,  the 
dissolved  substance  is  more  abundant  in 
the  surface;  but  if  the  surface-tension  of 
a  solution  increases  with  augmented  con- 
centration, the  concentration  is  less  in 
the  surface  than  in  the  interior. 

The  law  is  also  expressed  as  follows : 

A  dissolved  substance  is  adsorbed  if  it  lowers  the  surface- 
tension;  in  the  opposite  case  the  adsorption  will  be  "  negative." 
It  is,  however,  not  advisable  to  designate  as  adsorption,  the 
accumulation  of  dissolved  substance  at  the  surface  of  the  solvent. 

To  the  substances  which  lower  the  surface-tension  of  water 
belong  most  compounds  which  are  not  strong  electrolytes,  such 
as  alcohols,  glycerol,  fatty  acids,  etc.  When  the  surface-con- 
centration attains  a  certain  value  owing  to  the  tendency  of  these 
substances  to  collect  at  the  surface  of  their  aqueous  solutions, 
the  head  of  the  osmotic  pressure  is  held  in  equilibrium. 

Unlike  these  substances,  many  salts  increase  the  surface- 
tension  of  water  towards  air  and,  presumably,  also  towards  other 
media,  so  that  an  increase  of  the  concentration  at  the  surface, 
like  that  just  described,  cannot  then  take  place.  On  the  other 
hand,  it  must  be  pointed  out  that  D  o  n  n  a  n  and  Barker 
(Proc.  Roy.  Soc.,  A,  1911,  85,  557)  have  recently  found,  in  cer- 
tain cases,  values  for  the  surface-concentration  (adsorption) 
which  agree  in  order  of  magnitude  with  those  calculated  from 
G  i  b  b  s  '  s  equation. 

What  is  understood  by  the  surface-tension  of  a  solution 
must  be  more  strictly  defined.  Since  the  surface-tension  depends 
on  the  concentration  and  since  in  every  fresh  surface  a  con- 
centration is  gradually  attained  which  differs  from  that  inside 
the  liquid,  it  is  evident  that  a  newly-formed  surface,  which 
has  not  reached  a  condition  of  concentration-equilibrium  with 
the  interior  of  the  solution,  possesses  a  different  surface-tension 


PHYSICAL  PROPERTIES  OF   THE  ENZYMES  75 

from  one  already  in  stable  equilibrium.  The  latter  value  is 
suitably  termed  the  static  and  the  former  the  dynamic 
surface-tension. 

ADSORPTION 

Of  the  possible  cases  of  adsorption,  those  exhibited  at  the 
limiting  surface  between  a  solution  and  a  solid  body  are  of  the 
greatest  interest  in  this  connection.  Use  is  often  made  of  such 
adsorptions  in  the  study  of  enzymes. 

These  phenomena  have,  to  some  extent,  been  known  for 
a  long  time,  although  the  facts  have  only  recently  been  satis- 
factorily collated. 

Attempts  have  been  made,  especially  during  the  last  few  years, 
to  conceive  of  adsorption  as  a  capillary  phenomenon.  But  the. 
above  thermodynamical  law  showing  the  connection  between 
the  change  of  surface-tension  and  the  adsorption  has  up  to  the 
present  not  proved  very  fertile.  In  the  experimental  proof  it 
was  necessary,  except  in  one  special  case,  to  assume  that  the 
surface-tension  of  water-gaseous  space  proceeded  parallel  with 
that  of  water-adsorbent;  and  there  was  nothing  to  indicate  that 
this  was  the  case.  A  "  negative  adsorption "  which  should, 
according  to  the  above  theory,  occur  with  electrolytes,  has  never 
been  observed  at  the  surface  of  separation  between  salt  solutions 
and  solid  adsorbing  material  (H  a  g  g  1  u  n  d  ,  H.,  1910,  64,  294),, 

Quite  recently  Arrhenius  (Medd.  Nobel-Inst.,  1911,  2, 
7)  has  subjected  the  experimental  results  of  Miss  Frances 
Homfray,  A.  Titoff  and  G.  C.  Schmidt  to 
calculation. 

As  regards  the  influence  of  the  quantity  of  the  adsorbing 
material,  it  is  the  magnitude  of  its  surface  which  is  of 
the  first  importance ;  for  one  and  the  same  preparation  the  quantity 
of  substance  adsorbed  is,  under  similar  conditions,  proportional 
to  the  active  surface.  Of  especial  importance  is  the  experimental 
result  of  the  investigations  of  G.  C.  Schmidt  (Zeitschr. 
f  physikal.  Chem.,  1910,  74,  689),  namely,  that  the  quantity 
adsorbed  increases  only  to  a  maximum,  no  matter  how  high  the 
concentration  of  the  surrounding  solution  rises.  This  maximum 
is  proportional  to  the  amount  of  the  adsorbing  medium  and 
varies  with  its  nature. 


76  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

G.    C.    Schmidt    has  investigated  the  simplest  mathe- 
matical formulation  of  his  results,  namely, 

dx       * 


where  s  represents  the  maximum  adsorption,  x  the  quantity 
adsorbed,  and  c  the  concentration  of  the  surrounding  solution. 
But  the  integral  equation  derived  from  the  above  expression 
is  not  in  agreement  with  the  observations.  On  the  other  hand, 
the  formula  deduced  theoretically  by  Arrhenius, 

7  dx  _(s—x) 
K—^J  —  —     —  , 
dc        x 


holds  generally  for  adsorption  phenomena  at  low  temperatures. 
This  formula,  on  integration,  gives 

logio  —  -0-4343-  =7-  c. 

s  —  x  s     k 

Here  x  represents  the  amount  condensed  on  1  grm.  of  the 
adsorbing  medium  (charcoal),  s  the  maximum  value  of  this 
amount,  c  the  osmotic  pressure  of  the  solute  (or  the  pressure 
of  the  surrounding  gas  which  is  adsorbed),  and  k  a  constant. 

Since  the  value  of  s  is  determined  directly  from  the  observations, 
the  formula  contains  only  one  arbitrary  constant,  whilst  that 
previously  in  general  use,  namely, 

-  =  kcn, 
m 

contains  two,  k  and  n.  In  spite  of  this,  however,  the  new 
formula  agrees  much  better  with  the  observations. 

Another  empirical  formula  for  the  adsorption-equilibrium 
embracing  a  wider  region  was  given  by  Freundlich  (Zeitschr. 
f  .  physikal.  Chem.,  1907,  57,  385)  : 


v  i         a       ^        ia\ 

-log =X  =  a(-J 

m        a  —  x  \v  / 


In  this  expression  a  indicates  the  total  quantity  of  solute  and 
v  the  volume  of  the  liquid.     The  magnitude  X  is  independent 


PHYSICAL  PROPERTIES  OF  THE  ENZYMES  77 

of  the  quantity  of  adsorbing  substance,  but  is  a  function  of 
a  and  v,  or,  more  strictly,  of  the  ratio  between  them;  a  and  n 
are  magnitudes  depending  only  on  the  temperature  and  on  the 
nature  of  the  solute. 

The  adsorption-equilibrium  determined  by  the  given  formula 
must  be  completely  reversible  and  independent  of  the 
path  by  which  it  is  reached.  The  adsorbing  medium  charged 
with  adsorbed  substance  must  give  up  the  latter  to  the  pure 
solvent  until  a  new  equilibrium  is  attained. 

This  reversible  adsorption  is  often  followed  by  a  consequent 
phenomenon — the  fixing  of  the  adsorbed  substance — brought 
about  partly  by  a  change  of  this  substance  (which  may  become 
insoluble,  for  instance)  and  partly  by  a  chemical  reaction  with 
the  adsorbent — a  reaction  which,  in  many  cases,  leads  to  the 
destruction  or  denaturation  of  the  adsorbed  material.  Many 
colouring  matters  are  fixed  from  true  solutions,  but  this  more 
stable  union  takes  place  especially  with  colloids,  in  particular 
with  proteins,  toxines  and  enzymes.  This  fixation  is  n  o  n  - 
reversible.  With  the  toxines,  the  sum-total  of  the  phe- 
nomena of  antitoxine-formation  is  highly  involved  and  has  given 
rise  to  keen  controversies.  With  enzymes,  quite  analogous 
processes  are  known,  e.g.,  the  combination  of  trypsin  and  anti- 
trypsin,  and  the  fixing  of  various  enzymes  by  charcoal  ( S  . 
G  .  H  e  d  i  n ,  H.,  1907,  50,  497),  to  which  reference  will  be 
made  later. 

It  must  here  be  mentioned  that  all  adsorption  phenomena 
are  by  no  means  to  be  attributed  to  one  and  the  same  cause. 

Besides  the  mechanical  adsorptions  already  mentioned, 
there  exist  a  large  number  of  adsorption  phenomena  caused 
by  chemical  transformations,  and  this  is  especially  the 
case  with  acid  and  basic  dyes,  which  are  not  adsorbed  mechanically, 
but  combined  chemically,  by  animal  fibres.  L.  Michaelis 
has  repeatedly  pointed  out  the  chemical  nature  of  many  adsorp- 
tion processes. 

As  Michaelis  rightly  stated  (Oppenheimer's 
Handbuch,  II,  1,  390),  of  all  known  substances,  charcoal  and 
cellulose  are  the  only  ones  with  which  mechanical  adsorption 
occurs.  "  Almost  all  other  substances  known  as  adsorbents, 
such  as  silicic  acid,  kieselguhr,  kaolin,  arsenic  sulphide,  mastic, 
ferric  hydroxide,  clay  and  zirconium  hydroxide,  have  practically 


78  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

no  mechanical  adsorptive  power  and,  in  general,  do  not  adsorb 
electro-indifferent  substances  like  alcohol,  acetone  and  sugars. 
They  adsorb  only  substances  which  can  occur  in  the  form  of 
ions  or  of  electrically-charged  suspensions,  the  adsorption  taking 
place  only  in  accordance  with  their  electrical  charge.  Thus 
silicic  acid  adsorbs  only  such  substances  as  migrate  towards 
the  cathode,  and  ferric  hydroxide  only  those  migrating  to  the 
anode."  With  these  substances,  then,  it  is  always  a  question  of 
a  neutralisation  between  an  acid  and  a  basic  compound  at  the  sur- 
face of  the  adsorbing  medium.  According  to  M  i  c  h  a  e  1  i  s 
and  R  o  n  a  (Biochem.  Z.,  1908,  15,  196),  even  adsorption  by 
charcoal  is  not  always  purely  mechanical,  but  sometimes  takes 
place  by  means  of  electrical  forces. 

If  two  substances  are  adsorbed  from  a  solution  by  an  adsorp- 
tion medium,  they  may  replace  one  another  (Michaelis). 
This  fact  is  evidently  closely  related  toG.  C.  Schmidt's 
discovery  that  the  adsorption  reaches  a  maximum.  This  phe- 
nomenon plays  a  part  in  the  experiments  of  H  e  d  i  n  (H., 
1909,  63,  143),  who  found  that  the  retardation  of  rennet-action 
by  charcoal  is  counteracted  by  other  substances,  e.g.,  serum, 
white  of  egg,  etc. 

Colloids.  The  adsorption  phenomena  of  greatest  interest 
in  the  study  of  the  enzymes  are  those  in  which  colloids  take 
part.  On  the  one  hand,  these  substances  can,  in  the  solid  or 
gelatinised  condition,  function  as  adsorption  media,  and,  on 
the  other,  they  are  themselves  largely  adsorbed  by  solid  sub- 
stances. 

In  the  following  considerations,  we  may  limit  ourselves  to 
one  of  the  two  large  groups  into  which  colloids  are  divided, 
namely,  the  so-called  emulsion-colloids  or  emulsoids,  and  may 
omit  any  description  of  the  suspension-colloids  or  suspensoids 
which  play  virtually  no  part  in  enzymic  solutions  or  in  investiga- 
tions of  these. 

While  the  suspensoids,  to  which,  for  example,  colloidal  metals 
belong,  are  classed  with  the  true  macroscopic  suspensions,  the 
emulsoids  are  so  closely  related  to  the  crystalloids  that  no  sharp 
limit  can  be  drawn  between  them.  With  a  number  of  classes 
of  bodies,  increase  of  molecular  weight  is  accompanied  by  the 
appearance  of  a  tendency  to  form  complexes  and  thus  pass  into 


PHYSICAL  PROPERTIES  OF  THE  ENZYMES  79 

the  colloidal  state.  Good  examples  of  this  are  presented  by 
the  condensation  products  of  glucose — dextrin  and  starch — 
E.  Fischer's  polypeptides,  and,  according  toF.Krafft's 
investigations  (Chem.  Ber.,  1895,  28,  2566;  1896,  29,  1328) 
especially  the  fatty  acids.  Whilst  the  lower  fatty  acids  exhibit 
normal  ionisation  and  normal  osmotic  pressure,  sodium  laurate, 
Ci2H2s02Na,  for  example,  occurs  principally  in  doubled  molecules 
and  sodium  oleate,  CisHssCbNa,  in  20%  solution,  produces 
no  measurable  elevation  of  the  boiling  point  and  thus  behaves 
as  a  substance  of  infinitely  large  molecular  weight  (Krafft). 
The  variation  in  properties  is  hence  continuous  from  the  emulsion- 
colloids  to  the  crystalloids.  The  osmotic  pressure  and  the 
magnitudes  related  to  it  are  very  small,  even  on  the  basis  of 
the  simplest  possible  molecular  formula,  and  become  still  smaller 
with  the  increasing  tendency  to  complex-formation  accompanying 
increase  of  molecular  weight.  The  values  then  often  fall  within 
those  due  to  inefficient  methods  of  purification  or  within 
the  unavoidable  limits  of  error,  or  else  are  of  little  significance 
owing  to  the  material  employed  being  chemically  ill-defined. 
But  where  highly-condensed  substances,  such  as  starch,  glycogen, 
etc.,  can  be  obtained  in  a  state  of  considerable  purity,  the 
depressions  of  freezing  point  and  elevations  of  boiling  point 
indicate  molecular  weights  of  at  least  100,000,  these  values 
being  only  minimal  ones. 

As  regards  the  molecular  weights  of  the  enzymes — with  the 
exception  of  the  oxydases  of  Medicago  which  were  inves- 
tigated by  the  author  and  B  o  1  i  n  (H.,  1909,  61,  1)— nothing 
certain  is  as  yet  known.  Great  care  should  be  exercised  in 
drawing  conclusions  concerning  the  molecular  magnitudes  of 
enzymes  from  the  diffusion  experiments  of  R.  0.  Herzog 
and  Kasarnowski  (Zeitschr.  f.  Elektrochem.,  1907,  13, 
527;  Biochem.  Z.,  1908.  11,  172)  on  commercial  enzyme-prepa- 
rations, partly  on  account  of  the  very  small  enzyme-contents 
of  these  preparations  and  partly  owing  to  the  considerable 
weakening  to  which  they  must  have  been  subjected  during  the 
investigations.  With  one  of  their  purest  invertase  preparations, 
Euler  and  Kullberg  (H.,  1911,  73,  335)  have  made 
diffusion  experiments,  the  results  being  calculated  by  means 
of  the  author's  formula,  D\/M  =  const.  (Wied.  Ann.,  1897, 
63,  273).  For  the  coefficient  of  diffusion  at  17°  the  value  0-037 


80  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

was  obtained  and  this  gave — with  considerable  extrapolation, 
it  is  true — the  molecular  weight  as  27,000.  This  number  repre- 
sents a  minimal  value  and  holds  for  neutral  solution. 

With  substances  of  such  great  molecular  magnitudes, 
Brownian  movement  begins  to  become  visible.  This  move- 
ment is  shown  by  small  particles  suspended  or  dissolved  in  a 
colloidal  state  in  a  solvent  and  consists  of  a  continuous  irregular 
motion,  which  increases  in  rapidity  with  the  fineness  of  the 
suspended  substance  and  with  diminution  of  the  internal  friction 
of  the  liquid. 

This  motion  is  to  be  regarded  as  an  expression  of  the  general 
molecular  motion  of  matter,  as  has  been  assumed  by  the  kinetic 
theory  of  heat  since  the  middle  of  last  century.  Brownian 
movement  has  been  thoroughly  studied  in  the  case  of  suspension- 
colloids,  but  not  with  emulsion-colloids. 

In  the  ultramicroscope  of  Siedentopf  and  Z  s  i  g  - 
m  o  n  d  y  the  emulsoids  mostly  show  only  a  cone  of  diffused 
light,  i.e.,  the  particles  are  generally  too  small  to  be  perceived 
ultramicroscopically  as  discrete  forms. 

Particles  visible  in  the  microscope  have  diameters  down  to  about 
2.10~5  cm.  (microns).  As  submicrons  are  known  those 
particles  which  are  perceptible  only  in  the  ultramicroscope,  whilst 
those  the  existence  of  which  can  only  be  perceived  indirectly,  even  in 
the  ultramicroscope,  are  termed  amicrons  and  have  diameters  of 
from  5.10- 7  to  1.10~7  cm. 

Everything  goes  to  indicate  that  colloidal  solutions  are  to 
be  regarded  as  mixtures  of  larger  and  smaller  molecular  aggregates, 
which  are  able  to  change  into  one  another  with  greater  or  less 
rapidity.  The  most  active  chemically  must  always  be  the 
smallest  and  therefore  the  really  dissolved  parts,  which  explains 
why,  in  chemical  transformations,  colloidal  substances  follow 
the  laws  of  reaction  derived  theoretically  for  dissolved 
substances. 

The  surface-tension  of  water  is  very  considerably  depressed 
by  emulsion-colloids,  as  is  shown  by  qualitative  observation. 
According  to  Quincke  (Wied.  Ann.,  1888,  [iiil,  35,  580), 
the  value  of  a  for  a  10%  tannic  acid  solution  is  29%  less  than 
that  for  water;  the  surface-tension  of  a  dilute  gelatine  solution 
is  28%  less.  Consequently  these  substances  exhibit  a  marked 


PHYSICAL  PROPERTIES  OF  THE  ENZYMES  81 

tendency  to  concentrate  at  the  surfaces  of  the  solutions  and  are 
strongly  adsorbed. 

As  regards  the  first  phenomenon,  the  accumulation  of  the 
colloid  at  the  surface,  this  is  exercised  in  an  especially  striking 
manner  in  the  formation  of  solid  membranes  of  peptone  at  the 
surface  of  gelatinous  peptone,  either  with  or  without  some 
chemical  change. 

Just  as  clearly  is  the  concentration  of  emulsion-colloids  at  the  surface 
of  aqueous  solutions  seen  if  the  liquid  is  shaken  and  the  composition 
of  the  foam  examined.  Further,  the  great  "  head-retaining  "  properties 
of  many  solutions,  e.g.,  of  albumins,  constitute  an  indication  of  accu- 
mulation of  these  substances. 


SOLID,   NEUTRAL   ADSORPTION-MEDIA 

Charcoal  has  often  been  used  as  an  adsorbent  for 
enzymes.  Thus,  S  .  G  .  H  e  d  i  n  (Bio-chemical  Journ.,  1906, 
1,  484;  1907,  2,  81,  112;  H.,  1907,  50,  497)  showed  that  trypsin 
is  adsorbed  by  animal  charcoal;  if  a  sufficient  quantity  of  the 
latter  is  employed,  the  adsorption  is  complete.  Also,  a  t 
first  it  is  reversible.  But  later  the  process  which  has  been 
already  mentioned  and  is  not  uncommon  with  adsorbed,  organic 
substances,  viz.,  fixing,  takes  place.  Fixing  is  a  relatively 
slow  process  and  the  amount  of  trypsin  fixed  increases  with  the 
amount  of  animal  charcoal  and  with  rise  of  temperature.  Whilst, 
therefore,  water  is  no  longer  able  to  extract  the  fixed  trypsin 
from  the  charcoal,  casein  is  able  to  do  so  and,  the  higher  the 
temperature,  the  more  completely  is  this  the  case.  Charcoal 
and  talc  act  in  a  similar  manner  towards  rennet  (H  e  d  i  n,  H., 
1909,  60,  364),  which  can  also  be  removed  from  the  charcoal  by 
the  substrate. 

According  to  the  same  author,  the  a-  and  g-proteases  occurring 
in  ox-spleen  are  likewise  taken  up  by  animal  charcoal  in  similar 
proportions. 

In  agreement  with  H  e  d  i  n  's  results  are  those  of  E. 
Buchner  andF.  Klatte  (Biochem.  Z.,  1908,  9,  436), 
who  found  that  trypsin  is  adsorbed  from  dilute  solution  by  threads 
of  silk,  wool  and  cotton,  strips  of  linen,  paper  and  agar-agar 
and  by  asbestos  and  glass-wool. 


82  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Lipase  can  be  completely  removed  from  either  an  alkaline 
or  an  acid  solution  by  means  of  charcoal  or  kaolin  (L.  M  i- 
c  h  a  e  1  i  s  and  P.  R  o  n  a  ,  Biochem.  Z.,  1907,  4,  11;  L.  M  i- 
c  h  a  e  1  i  s,  ibid.,  1908,  7,  488.  See  also  L.  M  i  c  h  a  e  1  i  s  and 
M.  Ehrenreich,  ibid.,  1908,  10,  283).  , 

Especially  noteworthy  is  the  fact  that,  in  the  adsorption 
of  colloids  by  charcoal,  etc.,  the  colloids  exert  a  reciprocal  influence, 
and  that  even  crystalloids,  such  as  glucose,  may  diminish  the 
capacity  of  charcoal  to  take  up  other  crystalloids,  presumably 
by  altering  the  surface-tension. 

On  this  fact  depends  the  phenomenon,  studied  by  H  e  d  i  n 
(H.,  1909,  63,  143),  namely,  that  the  retardation  of  rennet-action 
produced  by  charcoal  is  prevented  by  various  substances. 

With  these  neutral  adsorption  media,  which  are  generally 
employed  as  powders,  must  be  classed  those  which  adsorb  when 
in  the  form  of  solid  layers,  such  as  cellulose  as  filter-paper  (on 
this  depend  G  r  u  s  s  's  investigations  [Bot.  Ber.,  1908,  26a, 
191  and  1909,  27,  313]  on  the  capillary  analysis  of  enzymes), 
and  the  materials  of  the  various  filter-candles,  e.g.,  the  Chamber- 
land-filter.  Their  behaviour  towards  enzymes  is  characterised 
principally  by  their  ability  or  inability  to  retain  the  enzymes 
when  solutions  of  the  latter  are  filtered.  Here,  too,  the  molec- 
ular magnitude  or  the  size  of  the  particles  of  the  substance  to  be 
filtered  comes  into  play,  the  filter  acting  not  only  as  an  adsorp- 
tion medium  but  directly  as  a  'sieve;  with  emulsion-colloids, 
however,  the  adsorption  is  usually  the  more  important  process. 
Of  great  interest  are  the  investigations  of  H  o  1  d  e  r  e  r  (C.  R.r 
1909,  149,  1153;  1910,  150,  230,  285  and  790),  who  showed  that 
the  permeability  of  the  Chamberland-filter  for  enzymes  depends 
on  the  concentration  of  the  hydrogen  ions  in  the  solution.  If 
the  solution  is  neutral  towards  phenolphthalein  (OH'  =  10~6), 
no  adsorption  takes  place,  the  filter  being  permeable;  but  if  the 
liquid  is  neutral  to  methyl  orange,  the  filter-candle  is  impermeable. 
This  is  found  to  be  the  case  with  invertase,  catalase,  pepsin  and 
emulsin.  The  following  data  show  the  behaviour  of  the  enzymes 
towards  the  most  common  filtering  materials. 


PHYSICAL  PROPERTIES  OF  THE  ENZYMES  83 


Chamberland-filter 

The  following  are  retained : 
Lipases  of  various   origins   (Fermi    and  Pernossi,    Ann. 

Inst.  Pasteur,  1889,  3,  531). 

Yeast-invertase  (Fermi    and    Pernossi,    ibid.)- 
Zymase   (B  u  c  h  n  e  r,    Zymasegarung,  Munich,   1903). 
Pepsin  and  trypsin. 

The  following  pass  through : 

Maltase   (Croft  Hill,    Journ.   Chem.   Soc.,   1898,  73,  636). 
Stomach-steapsin   (V  o  1  h  a  r  d  ,    Zeitschr.   f.   klin.   Med.,  1901, 

42,  414). 

Liver-aldehydase  (J  a  c  o  b  y,   H.,  1900,  30,  135). 
Proteinases  of  malt  (Fernbach   and  Hubert,  C.  R.,  1900, 
130,  1783;   131,  293). 

Parchment 

These  are  retained: 

Pepsin  (Hammarsten,  Maly's  Jahresber.,  1874,  3,  160). 
Peroxydase  (E  u  1  e  r  and  B  o  1  i  n  ,   H.,  1909,  61,  82.) 

These  pass  through : 
Invertin,    amylase,    and,    slowly,    emulsin,    trypsin  and    pepsin 

(Chodschajew,   Arch,  de  Physiol.,  1898,  30,  241). 
Rennet  and  pepsin  pass  through  unstretched  amnion-membrane 

(J  a  c  o  b  y,  Biochem.  Z.,  1906,  1,  53). 
Rennet,  invertin  and  catalase  pass  through  intestinal  membrane 

(Vandevelde,  Biochem.  Z.,  1906,  1,  408). 
Rennet,   invertin   and   catalase   do   not   pass   through   cellulose 

walls    (thimbles  from  L  e  u  n  e  ,   Paris)    (Vandevelde, 

loc.  cit.). 

Collodion-membranes 

The  following  is  retained : 
Pepsin  (S  t  r  a  d  a  ,   Ann.  Inst.  Pasteur,  1908,  22,  982). 

The  following  pass  through: 
Emulsin    and    lactase    (B  i  e  r  r  y     and     Schaeffer,     Soc. 

Biol.,  1907,  62,  723). 

Trypsin    partially:   after    activation    with    kinase,    completely 
(S  t  r  a  d  a  ,   1  o  c.    cit.). 


84  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Since  colloids  in  the  sol  condition  do  not,  in  general,  diffuse 
through  animal  membranes  and  colloidal  skins,  very  different 
membranes  may  be  employed  as  colloid-filters. 

In  the  gradation  of  the  colloid-content,  the  permeability  of  a  filter 
may  be  varied  at  will  by  employing  a  substratum  of  cellulose,  etc.,  so 
that  the  solution  may  be  fractionally  filtered.  On  this  principle  B  e  c  h  - 
hold  constructed  his  ultra-filter,  in  which,  as  colloids,  acetic  collodion 
and  gelatine  are  especially  used  (Zeitschr.  f.  physikal.  Chem.,  1907,  60, 
257;  1908,  64,  328). 

Fibrin  flocks  have  proved  effectual  for  the  adsorption  of 
many  enzymes,  such  as  rennet,  pepsin  (J  a  c  o  b  y,  Biochem. 
Z.,  1907,  4,  21)  and  trypsin  (B  u  c  h  n  e  r  and  K  1  a  1 1  e,  Bio- 
chem. Z.,  1908,  9,  436).  Numerous  other  coagulated  proteins 
also  exhibit  marked  adsorptive  properties.  (See  also  B  a  y  1  i  s  s  , 
Adsorption  in  its  relation  to  enzyme  action,  Kolloid.  Zeitschr., 
1908,  3,  224.) 

Solid  acid  or  basic  constituents.  The  chief 
of  these  are,  on  the  one  hand,  the  hydroxides  of  ferric  iron, 
aluminium  and  magnesium,  and  certain  of  the  so-called  colloid 
metals  (Rona;  Deleano,  Biochem.  Z.,  1909,  19,  266),  and, 
on  the  other,  silicic  acid.  In  this  case,  the  principal  action  is  a 
chemical  union  and  not  mechanical  adsorption;  this  is  shown 
by  the  selective  adsorption  of  these  sols,  acid  substances  being 
vigorously  adsorbed  by  the  metallic  hydroxides  and  basic  ones 
by  silicic  acid;  the  same  regularity  appears  in  the  reciprocal 
coagulation  of  the  colloids. 

Similar  influences  govern  the  adsorption  of  enzymes.  As 
M  i  c  h  a  e  1  i  s  found  (Biochem.  Z.,  1907,  7,  488),  electro- 
negative colloid  solutions  give  with,  say,  invertin  no  precipita- 
tion, whilst  the  hydroxides  of  iron  and  aluminium  completely 
adsorb  this  enzyme.  Analogous  behaviour  is  shown  by  pepsin 
(Biochem.  Z.,  1908,  10,  283).  On  the  other  hand,  the  adsorp- 
tion of  amylase  and  saliva-diastase  (ptyalin)  depends  on  the 
reaction  of  the  medium.  Zymase  appears  to  be  a  neutral  sub- 
stance (M  i  c  h  a  e  1  i  s  and  Rona)  and  is  consequently  not 
adsorbed  by  ferric  hydroxide;  but  the  co-enzyme  of  zymase 
seems  to  be  readily  adsorbed  by  ferric  hydroxide  (R  e  s  e  n- 
s  c  h  e  c  k,  Biochem.  Z.,  1908,  15,  1). 

To  the  acid  adsorption  media  belongs   also  kaolin,   which 


PHYSICAL  PKOPERTIES  OF  THE  ENZYMES  85 

hence  adsorbs  mainly  basic  substances.  Since  the  rule,  that 
acid  and  basic  adsorption  media  adsorb  respectively  basic  and 
acid  substances,  has  proved  generally  valid,  we  are  able,  from 
observations  on  adsorption,  to  draw  conclusions  concerning 
the  electro-chemical  nature  or  the  charge  of  adsorbable  substances, 
in  particular  of  the  enzymes.  These  conclusions  are  remarkably 
well  confirmed  by  other  facts. 

Electric  transference.  Under  the  influence  of 
a  difference  of  electric  potential,  emulsion-colloids  migrate  in 
the  same  manner  as  particles  suspended  in  water;  this  effect 
is  known  as  cataphoresis.  But,  whilst  suspended  particles 
migrate,  as  a  rule,  to  the  positive  pole  and  themselves  assume  a 
negative  charge,  (according  to  C  o  e  h  n  '  s  rule,  this  behaviour 
depends  on  the  fact  that  the  dielectric  constant  of  these  particles 
is  less  than  that  of  water),  emulsion-colloids  migrate  partly  to 
the  positive  and  partly  to  the  negative  pole.  This  is  com- 
prehensible if  it  is  borne  in  mind  that  emulsion-colloids  have, 
as  a  rule,  basic  or  acid  properties,  or — as  amphoteric  electrolytes 
— may  exhibit  the  one  or  the  other  character  according  to  the 
medium  in  which  they  exist.  As  with  the  ions,  the  charge  which 
they  assume  on  ionisation  determines  the  direction  of  migration. 
Further,  the  velocity  of  migration  is  not  appreciably  different 
from  those  of  dissolved  ions;  according  to  Whitney  and 
Blake,  for  gelatine  particles  it  has  the  value 

25.10 ~5  cm./sec. 

for  1  volt /cm.,  the  corresponding  value  for  sodium  ions  being 
43.10~°.  On  the  other  hand,  in  the  cataphoresis  of  emulsion- 
colloids  there  appear  various  marked  disturbances,  caused  partly 
by  the  migration  of  the  particles  from  the  two  electrodes  and  their 
precipitation  in  the  medium  as  oppositely  charged  sols. 

How  great  is  the  dependence  of  the  electric  transference 
of  proteins  on  the  reaction  of  the  medium  is  best  seen  from 
Pauli's  researches  (Hofm.  Beitr.,  1906,  7,  531).  Albumin 
poor  in  electrolytes  shows  no  motion  under  a  pressure  of  250 
volts,  whilst  even  inO  -01  N-hydrochloric  acid  it  assumes  an  electro- 
positive, and  in  dilute  alkali  a  negative  character. 

P  a  u  1  i  and  Handovsky  (Biochem.  Z.,  1909,  18,  340) 
and  also  M  i  c  h  a  e  1  i  s  have  recently  subjected  these  questions 
to  a  fresh  and  thorough  investigation. 


86  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

The  most  important  results  concerning  the  electric  trans- 
ference of  enzymes  are  due  to  M  i  c  h  a  e  1  i  s.  He  found  firstly 
(Biochem.  Z.,  1909,  16,  81)  that,  independently  of  the  reaction 
of  the  medium,  invertin  migrates  distinctly  to  the  anode 
and  is  hence  decidedly  acid  in  nature.  Pepsin  also 
exhibits  a  strong  negative  character  as,  in  neutral  and  even  in 
markedly  acid  solution,  it  migrates  solely  to  the  anode.  Shortly 
afterwards  (Biochem.  Z.,  1909,  17,  831),  it  was  also  found  possible, 
with  a  concentration  of  hydrochloric  acid  exceeding  5VN,  to 
obtain  electric  transference  of  pepsin  to  the  cathode,  agreement 
with  the  adsorption  analysis  being  therefore  complete.  On  the 
other  hand,  trypsin  and  diastase  behave  also  in  the  electric  field 
as  amphoteric  substances,  migrating  to  the  anode  or  cathode 
according  to  the  reaction  of  the  solution;  diastase  is,  however, 
more  strongly  positive  and  trypsin  more  strongly  negative. 

Michaelis  (Biochem.  Z.,  1909,  19,  181;  1910,  28,  1) 
has  determined  the  "  relative  acidity''  of  certain 
enzymes.  If 

Xa[undissociated  albumin]  =  [H  +  ]  [  Alb  ~  ] 
and 

X6[undissociated  albumin]  =  [OH  ~  ]  [  Alb  +  ]  , 

then,  in  an  iso-electric  state,  i.e.,  when  equal  members  of  positive 
and  negative  albumin-ions  are  present, 


K&     [OH-]' 
This  quotient  assumes  the  following  values: 

Malt-amylase,  1  Trypsin,  105-108 

Serum-albumin,  102  -  103  Pepsin,  5.  109 

Yeast-invertase,  oo  . 

The  relation  between  the  "  iso-electric  constant,"  /,  and  the 
relative  acidity,  R,  is  expressed  by  the  equation 

*^W 

where  kw  is  the  dissociation  constant  of  water. 

For  a  very  pure  pepsin,  Pekelharing  and  Ringer 
have  recently  (H.,  1910,  75,  282)  established  the  iso-electric 
point. 


PHYSICAL  PROPEKTIES  OF  THE  ENZYMES  87 

Bierry,  V.  Henri  and  Schaeffer  have  also  carried 
out  experiments  on  the  transference  of  enzymes — with  dialysed 
enzyme  solutions  (Soc.  BioL,  1907,  63,  226;  Biochem.  Z.,  1909, 
16,  473).  The  enzymes  investigated  were:  amylases  of  animal 
and  vegetable  origin,  invertin  from  yeast  and  from  Helix 
p  o  m  a  t  i  a,  emulsin  from  almonds  and  from  Helix  pomatia, 
lactase  from  Helix  pomatia,  rennet  (H  a  n  s  e  n  's)  and 
catalase  from  the  liver.  Only  one  of  these  enzymes,  namely, 
pancreas-amylase,  migrated  to  the  cathode,  all  the  rest  going  to 
the  anode. 

Even  in  0-01N-sodium  chloride  solution,  albumin  assumes 
an  electro-positive  character,  whilst  in  dilute  alkali  it  shows 
electro-negative  behaviour.  According  to  P  a  u  1  i 's  original 
results,  albumin  poor  in  electrolytes  shows  no  migration;  but 
more  detailed  investigations  by  P  a  u  1  i  and  Handovsky 
(Biochem.  Z.,  1909,  18,  340)  and,  especially,  by  M  i  c  h  a  e  1  i  s 
(ibid.,  1909,  19,  181)  show  that  neutral  albumin  or  albumin  at 
the  iso-electric  point — with  an  acidity  of  [H]  =  about  10  ~6 — does 
not  remain  stationary  but  migrates  to  both  the  anode  and  the 
cathode  at  the  same  time. 

Literature:  Michaelis,  Dynamik  der  Oberflachen, 
Dresden,  1909. 

The  precipitating  action  of  salts  on  the  colloids  has  been 
studied  in  great  detail,  especially  as  regards  the  influence  of 
alkali  salts  on  the  proteins. 

The  fundamental  investigations  on  this  subject  and  the 
application  of  precipitation  with  salts  to  the  fractionation  of 
mixtures  of  proteins  are  due  to  Hofmeister,  who,  as  early 
as  1887,  arranged  the  salts  in  the  order  of  their  precipitating 
actions.  His  work  has  been  considerably  extended  in  more 
recent  times  by  W  o.  P  a  u  1  i  (Hofm.  Beitr.,  1902,  3,  225;  1903, 
5,  27;  1905,  6,  233;  1906,  7,  531;  Biochem.  Z.,  1909,  17,  235, 
etc.). 

It  has  been  found  that  the  precipitating  properties  of  the  salts 
are  composed  additively  of  the  actions  of  the  cathions  and  anions. 
The  following  series  begins  with  the  ion  showing  the  greatest 
precipitating,  or  the  least  dissolving  power : 

S04,  HP04,  CH3C02,  Cl,  N03,  Br,  I,  CNS 
Li,  Na,  K,  NH4. 


88  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

With  the  exception  of  LiCl,  this  series  agrees  perfectly  with 
the  one  given  below  which  was  obtained  by  the  author 
(Zeitschr.  f.  physikal.  Chem.,  1899,  31,  360  and  1904,  49,  303) 
for  the  salting-out  of  non-electrolytes  ;  the  table  gives  the  mean, 
relative,  molecular  depression  of  solubility  (\w—  \s):  L,  where 
\w  is  the  solubility  of  the  non-electrolyte  in  water  and  L  its  solubility 
in  a  salt  solution  of  normal  concentration. 


NH4NO3  ........  0  KC1  ...........  0-23  iZnSO*  .........  0-31 

KI  .............  0-02  iBaCl2  .........  0-24  |K2SO4  .........  0-32 

KBr  ...........  0-05  |CaCl2  .........  0-24  |Na2SO4  ........  0-35 

KNO3  ..........  0-08  NaCl  ...........  0-25  £Na2CO3  ........  0-36 

NaNO3  .........  0-10  f(NH4)2SO4  ......  0-29  NaOH  .........  0-36 

LiCl  ...........  0-21  |MgSO4  .........  0-31 

It  is  therefore  beyond  doubt  that  the  same  phenomenon 
is  being  dealt  with  in  the  two  cases.  It  will  be  especially  seen 
that  ammonium  and  magnesium  sulphates,  which  are  most 
frequently  used  for  the  precipitation  of  proteins  and  also  of 
enzymes,  are  likewise  active  towards  crystalloids. 

The  remarkable  fact  that  the  rapidity  with  which  the  electro- 
lyte is  added  influences  the  completeness  of  the  precipitation,  has 
been  subjected  to  detailed  study  by  Freundlich  and 
by  H  6  b  e  r. 

If  small  quantities  of  an  acid  or  an  alkali  are  added  to  the 
protein  solution,  the  precipitating  actions  of  the  salts  are  modified 
and  the  order  of  the  ions  changed  (Posternak,  Ann.  Inst. 
Pasteur,  1901,  15,  85,  169,  570).  The  precipitation  of  proteins 
by  the  salts  of  the  alkaline  earth  metals  also  exhibits  peculiarities, 
which  have  been  studied  by  P  a  u  1  i  (Hofm.  Beitr.,  1903,  5,  27). 
Unlike  the  precipitations  produced  by  alkali  metals  or  magnesium, 
those  effected  by  salts  of  the  alkaline  earths  are  irreversible  ; 
they  differ  however,  distinctly  from  those  brought  about  by 
heavy  metals.  Further,  in  strongly  acid  solutions,  especially 
on  addition  of  alkali  salts,  irreversible  precipitations  occur. 

Especially  marked  is  the  effect  of  addition  of  acids  on  the 
action  of  neutral  salts  on  protein  When  a  little  acid  is  added 
to  carefully-dialysed  serum-albumin,  not  only  does  the  latter 
become  capable  of  migrating  to  the  negative  pole,  but  its  coagulabil- 
ity by  heat  and  by  alcohol  is  impaired.  At  the  same-  time  its 
viscosity  undergoes  considerable  increase.  Excess  of  acid, 
however,  restores  the  precipitability  by  alcohol  and  lowers  the 


PHYSICAL  PROPERTIES  OF  THE  ENZYMES  89 

viscosity  again.  Addition  of  any  neutral  salt  has  the  same  effect 
as  excess  of  acid  in  restoring  the  coagulability  of  acid-albumin 
by  heat  or  alcohol  and  in  diminishing  the  viscosity. 

The  emulsion-colloids  are  distinguished  by  the  considerably 
greater  internal  friction  of  their  solutions  from  the  pseudo- 
solutions  of  the  suspension-colloids,  which  often  possess  vis- 
cosities only  slightly  higher  than  that  of  the  pure  medium.  Very 
small  additions  of  salts  or,  more  especially,  of  acids  and  bases, 
produce  marked  changes  in  the  viscosity  of  proteins  and  the  con- 
clusion must  be  drawn  that  protein-  ions  give  rise  to  greater 
internal  friction  than  amphoteric  protein. 

The  course  of  proteolysis  has  often  been  followed  by  measuring  the 
viscosity  without,  however,  the  parallelism  between  the  composition  of 
the  solution  and  its  viscosity  being  sufficiently  clearly  proved. 

Jellies.  Many  colloid-containing  liquids  which,  to  indi- 
cate their  consistency,  are  termed  sols,  solidify  on  addition 
of  salts,  alcohol,  etc.,  to  an  apparently  homogeneous  mass  of 
peculiar  semi-solid  consistency — a  so-called  jelly.  The  best 
examples  are  silicic  acid  and  alumina  among  the  suspension- 
colloids  and  agar-agar  and  gelatine  among  the  emulsion-colloids. 
The  homogeneity  is,  however,  only  apparent.  Although  by 
no  means  in  all  cases,  yet  in  many  it  can  be  seen  under  the  micro- 
scope that  the  solidification  is  accompanied  by  a  de-mixing. 
The  course  of  this  change  has  been  followed  microscopically  by 
Hardy.  But  no  sharp  limit  exists  between  sols  and  gels  and 
P  a  u  1  i  was  right  when  he  emphasised  the  fact  that  all  grada- 
tions exist  between  solid  and  liquid  jellies,  and  that  a  jelly  is 
nothing  but  a  thick  sol  (Biochem.  Z.,  1909,  18,  367). 

According  to  P  a  u  1  i  (Hofm.  Beitr.,  1902,  2,  1),  salts  or  ions 
exert  the  same  relative  influence  on  precipitation  as  on  gelatinisa- 
tion,  so  that  the  series,  864 — Cl — CNS,  given  above  holds  also 
for  the  melting-  or  solidifying-point  of  gelatine.  A  corre- 
sponding parallelism  had  formerly  been  observed  by  H  o  f- 
m  e  i  s  t  e  r  between  precipitation  and  swelling. 


CHAPTER  III 

ACTIVATORS    (CO-ENZYMES),    PARALYSORS   AND 
POISONS 

FOR  the  occurrence  of  enzyme  reactions,  activators  or 
co-enzymes  are  undoubtedly  of  greater  and  more  general  impor- 
tance than  has  been,  until  quite  recently,  supposed.  In  many 
cases,  enzymic  processes  do  not  take  place  without  the  help  of 
specific  co-enzymes,  which,  indeed,  always  exert  a  considerable 
influence  on  the  course  of  the  reaction;  so  that  those  chemical 
substances  which  effect  the  activation  or  inactivation  of  the 
enzymes  must  be  studied  qualitatively  and  quantitatively  in 
as  complete  a  manner  as  possible.  Sorensen  and  also 
Hudson  have  recently  emphasised  the  marked  influence 
of  acids  and  bases,  or  of  the  concentrations  of  H+  or  OH~,  on 
enzyme  action,  and,  by  very  complete  investigations  on  invertase, 
catalase  and  pepsin,  have  obtained  numerical  expression  of  this 
influence.  As  regards  the  less  general  but  none  the  less  important 
influence  of  neutral  salts  and  non-electrolytes,  no  such  com- 
prehensive study  has  been  made,  so  that  a  resume  of  the 
numerous  qualitative  data  must  suffice. 

The  term  co-enzyme  has  been  applied  to  a  number  of  sub- 
stances which  take  part  in  enzymic  reactions.  But  this  name, 
which  has  come  into  very  general  use,  is  not  quite  suitable, 
since  it  characterises  the  substances  as  enzymes,  whilst  it  refers 
partly  to  inorganic  and  partly  to  organic,  thermostable  bodies 
of  known  compositions.  It  is  therefore  best  to  use  the  term 
"  activator  "  for  all  substances  which,  specifically  or  otherwise, 
participate  with  an  enzyme  in  the  acceleration  of  a  reaction. 

Following  the  ordinary  conception  and  method  of  nomenclature 
a  distinction  must  be  drawn  between  those  bodies  which  convert 
"  zymogens  "  into  the  active  state — kinases — and  those  which, 
on  the  other  hand,  intensify  enzyme  actions.  It  may  be,  however, 

90 


ACTIVATORS,  PARALYSORS  AND  POISONS  91 

as  will  be  mentioned  later,  that  kinases  do  not  act  in  an  essen- 
tially different  manner  from  other  activators. 


KINASES   OF  UNKNOWN  COMPOSITION 

As  is  well  known,  tryptase,  as  it  occurs  in  the  pancreatic 
juice,  is  activated  by  a  constituent  of  the  intestinal  liquid — 
enterokinase  1  (P  a  w  1  o  w  and  collaborators,  1900).  Although 
enterokinase  is  regarded  by  various  investigators,  especially 
B  a  y  1  i  s  s  and  Starling  (Journ.  of  Physiol.,  1904,  32,  129) 
and  Z  u  n  z  ,  as  an  enzyme,  Hamburger  and  H  e  k  m  a 
(J.  de  Physiol.  et  Pathol.  gen.,  1902  and  1904)  have  adduced 
important  evidence  in  support  of  the  view  that  the  formation 
of  trypsin  does  not  consist  in  a  new  enzyme  action  on  trypsinogen, 
but  that  a  definite  quantity  of  enterokinase  can  activate  only 
a  certain  amount  of  pepsinogen. 

The  preparation  of  pure  enterokinase  was  attempted  by 
Delezenne  (Soc.  Biol.,  1901,  53;  1902,  54).  It  can  be 
precipitated  from  intestinal  juice  by  calcium  phosphate  or  alcohol. 
Also  flocculent  fibrin  and  red  blood-corpuscles  adsorb  the  kinase, 
the  former  quantitatively.  Unlike  most  activators,  enterokinase 
is  n  o  t  stable  to  heat,  as,  according  to  Hamburger  and 
H  e  km  a  (loc.  cit.),  it  is  destroyed  at  67°  in  less  than  3  hours; 
but  B  i  e  r  r  y  and  Henri  (Soc.  Biol.,  1902,  54,  667)  assert 
that  it  retains  its  activity  after  being  heated  to  120°  for  20 
minutes. 

Enterokinase  dissolves  in  90%  alcohol  (Cohnheim,  Arch. 
Sci.  Biol.,  St.  Petersburg,  1904,  9,  Suppl.,  112). 

Kinases  which  activate  trypsinogen  were  found  by  H  o  n  - 
g  a  r  d  y  (Arch,  internat.  de  Physiol.,  1906,  3,  360)  in  milk  and 
by  Delezenne  in  leucocytes,  bacteria  and  fungi  and  also 
in  fibrin  (Soc.  Biol.,  1903,  55,  27  and  132). 

According  to  Morawitz  (Hofm.  Beitr.,  1903,  4,  381; 
1904,  5,  133)  and  others  (for  the  literature,  see  Buckmaster, 
Science  Progress,  1907,  2,  51),  a  kinase  of  unknown  character 
plays  a  part  in  the  formation  ofthrombin;  it  converts 
thrombogen  into  a-prothrombin,  which,  in  its  turn,  is  changed 
into  thrombin  by  lime. 

1  For  its  preparation,  see  B  a  y  1  i  s  s  ,  Journ.  of  Physiol.,  1904,  30,  80. 


92  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Thrombokinase  is  not  stable  to  heat.  According  to  investiga- 
tions by  E.  W.  A.  Walker  (Proceedings  of  the  Physiol. 
Soc.,  Dec.  16,  1905,  see  Journ.  of  Physiol.,  1905,  33,  xxi)  coagu- 
lated oxalate-plasma,  which  has  been  heated  at  50°  for  2 
hours,  coagulates  n  o  t  on  addition  of  calcium  chloride  alone, 
but  when  blood  or  fresh  tissue-extract  is  added  at  the  same  time. 
So  that  a  thrombogen  would  be  stable  at  50°,  whilst  thrombo- 
kinase  is  destroyed  at  this  temperature. 

Walker  found  that  saliva-amylase  which  had  been 
inactivated  by  heating  at  50-55°  could  be  re-activated  by  blood 
and  he  therefore  regarded  this  as  the  mutual  action  of  a  thermo- 
stable enzymogen  and  a  kinase  sensitive  to  heat. 

Further  investigation  of  these  phenomena  is  to  be  desired. 

In  most  other  cases  e.g.,  in  that  of  the  esterases  in  which  the 
existence  of  "  kinases  "  has  been  assumed  from  the  results  of 
experiments  with  artificially  inactivated  enzymes,  it  is  the  action 
of  thermostable  activators  which  has  been  observed. 

That  Rosenheim  succeeded  in  separating  the  lipase  of 
the  pancreas  from  an  activator  by  filtration,  has  been  already 
mentioned  (p.  10). 

The  composition  of  the  organic  activator  s  t  i  m  u  1  i  n, 
mentioned  by  Danilewski  and  more  closely  examined  by 
Schapirow,  is  also  unknown. 

Cohnheim  has  recently  found  that  an  extract  can  be 
obtained  from  the  boiled  pancreas  of  the  cat  which  strongly 
activates  the  glycolytic  enzyme  of  the  muscles.  Nothing  is, 
however,  known  concerning  the  chemical  nature  of  this  interest- 
ing activator.  It  is  precipitated  by  phosphotungstic  acid 
(Hall,  Amer.  Journ.  of  Physiol.,  1907,  18,  283). 

SPECIAL   ORGANIC   ACTIVATORS 

In  the  case  of  pancreas-lipase,  the  chemical  nature  of  an 
organic  activator  has  recently  been  established.  According 
to  the  observations  ofNencki,  Pawlow  and  Bruno, 
Rachford,  Magnus,  and  Loevenhart  (Journ.  of 
Biol.  Chem.,  1907,  2,  391),  bile  intensifies  the  hydrolysis  of 
fats  by  pancreatic  juice;  still  greater  effects  were  found  by 
D  o  n  a  t  h  (Hofm.  Beitr.,  1907,  10,  390)  to  be  produced  by  salts 
of  the  bile  acids.  These  salts  are  contained  in  the  co-enzyme, 


ACTIVATORS,  PARALYSORS  AND  POISONS  93 

resistant  to  boiling,  obtained  by  R.  M  a  g  n  u  s  (H.,  1902,  42, 
149)  from  the  liver.  Lecithin  has  no  action  (Kalaboukoff 
and  T  e  r  r  o  i  n  e,  Soc.  Biol.,  1907,  63,  617)  or  only  a  slight  one 
(Loevenhart  and  Sou  d  e  r,  Journ.  of  Biol.  Chem.,  1907, 
2,  415).  A  similar  intensifying  action  is  produced  by  the  sodium 
salts  of  the  synthetic  glycocholic  and  taurocholic  acids.  The 
co-enzyme  can  be  separated  from  liver-lipase  by  dialysis.  It 
must  be  emphasised  that  these  substances  accelerate  the  action 
of  pancreas-lipase  specifically  and  have  no  action  on  the  lipase 
of  the  stomach  (L  a  q  u  e  u  r,  Hofm.  Beitr.,  1906,  8,  281)  or  on 
that  of  the  intestines  (Boldyreff,  Zentralbl,  f.  Physiol., 
1904,  18,  460;  H.,  1907,  50,  394). 

Of  some  importance  is  the  observation  of  O.  Rosenheim 
and  Shaw-Mackenzie  (Journ.  of  Physiol.,  1910,  40) 
that  substances  which  exert  a  hsemolytic  action,  such  as  alcohol, 
soaps,  saponins,  digitoxin,  increase  the  action  of  pancreas- 
lipase.  Such  activation  is  annulled  by  cholesterol.  The  hydro- 
lytic  action  of  pancreas-lipase  is  augmented  also  by  blood- 
serum. 

The  action  of  the  amylases  is  also  intensified  by  bile  salts 

(Wohlgemuth,    Biochem.  Z.,   1909,  21,  447).     The  action 

of  these  salts  on  the  amylase  of  the  pancreas  is,  as  was  pointed 

•  out  by  Buglia    (Biochem.  Z.,  1910,  25,  239),  independent  of 

the  concentration  of  the  enzyme. 

That  bile  contains  also  an  activator  for  trypsin  has  been 
long  known  (Rachford,  Journ.  of  Physiol.,  1899,  25,  165; 
Delezenne,  Soc.  Biol.,  1902,  54,  283;  von  Fiirth  and 
Schtitz,  Hofm.  Beitr.,  1906,  9,  28;  Wohlgemuth, 
Biochem.  Z.,  1906,  2,  264). 

It  is,  however,  doubtful  whether  bile  salts  represent  a  specific 
kinase,  as  it  may  be  that  they  influence  the 
condition  of  solution  of  the  substrate.  Men- 
tion must  be  made  of  D  o  n  a  t  h  's  view — that  bile  salts  do 
not  activate  the  ready-formed  lipase,  but  accelerate  the  conver- 
sion of  the  lipase-zymogen  into  the  enzymic  state  (Hofm.  Beitr., 
1907,  10,  390). 

Amino-acids  are  noteworthy  as  activators  of  amyl- 
ase (see  Chapter  IV;  also  Ford  and  G  u  t  h  r  i  e  ,  Journ. 
Chem.  Soc.,  1906,  89,  76;  Journ.  Inst.  Brewing,  1908,  14,  61). 
Pancreatic  juice  is  also  activated  by  amino-acids.  W  o  h  1  g  e- 


94  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

m  u  t  h    (Biochem.  Z.,   1906,   2,  264)  observed   this  effect  with 
glycocoll,  alanine  and  other  representatives  of  this  group. 

According  to  R  e  i  c  h  e  1  and  S  p  i  r  o  (Hofm.  Beitr.,  1905, 
7,  504),  lecithin  accelerates  the  action  of  rennet.  Of  much 
greater  importance  is  the  activation  to  which  z  y  m  a  s  e  is 
subjected  under  the  influence  of  lecithin  and  other  organic 
compounds  of  phosphorus.  The  investigations  of  Harden, 
Young,  and  B  u  c  h  n  e  r  and  Meisenheimer  have 
shown  that  these  phosphorus  compounds  constitute  the  active 
constituent  in  boiled  pressed  yeast-juice  (compare  p.  54  and 
Chapter  IV). 

ACIDS,  BASES  AND   NEUTRAL   SALTS 

Between  the  actions  of  purely  specific  activators  or  catalysts 
and  the  general  actions  of  acids  and  bases  a  definite  limit  can 
hardly  be  drawn.  Thus,  according  to  H  o  y  e  r  '  s  investigations 
(H.,  1906,  50,  414),  the  action  of  lactic  acid  on  the  lipase  of  the 
castor-oil  seed  can  also  be  produced  by  other  acids,  the  action 
being,  however,  greater  than  corresponds  with  the  degree  of 
dissociation  of  this  specific  "  seed-acid."  Similar  relations  are 
observed  with  pepsin. 

Apart  from  the  activation  of  the  zymogens,  acids  and  bases 
can  influence  enzymic  reactions  in  two  different  ways,  which 
must  be  clearly  distinguished :  firstly,  the  velocity  of 
the  reaction  is  changed  and  reaches  a  well-defined  max- 
imum for  a  certain  concentration  of  the  hydrogen-ions;  secondly, 
acids  and  bases  influence  the  stability  or  the  decom- 
position of  the  enzyme  itself,  the  stability  also 
exhibiting  a  maximum  for  a  certain  concentration  of  the  H4" 
or  OH  ~  ions. 

Pepsin  requires  the  presence  of  a  free  acid  as  an  absolutely 
necessary  activator.  Pepsin  solutions  are,  indeed,  proteolytically 
active  only  when  they  contain  positive  pepsin-ions.  In  the 
organism  it  is  the  hydrochloric  acid  which  converts  the  pepsin- 
forming  secretion  of  the  mucous  membrane  of  the  stomach— 
L  a  n  g  1  e  y  's  "  pepsinogen  "  or  pro-pepsin — into  the  active 
enzyme. 

Numerous  investigations  have  been  made  on  the  replacemene 
of  the  hydrochloric  acid ;  of  the  older  ones,  those  of  P  f  1  e  i  - 


ACTIVATORS,  PARALYSORS  AND  POISONS  95 

d  e  r  e  r  (Pfliig.  Arch.,  1897,  66,  605),  von  Moraczewski, 
H  a  h  n  (Virch.  Arch.,  1894,  137,  597)  and  S  j  6  q  u  i  s  t  (Skand. 
Arch.  f.'Physiol.,  1895,  5,  277)  may  be  mentioned.  The  following 
tables,  from  a  paper  by  L  a  r  i  n  (Biochem.  Zentralbl.,  1905,  1, 
484),  and  from  that  of  Sjoquist,  give  the  acids  arranged  in  the 
order  of  magnitude  of  their  accelerating  action. 

Larin  Sjoquist 

1.  Hydrochloric  acid  7.  Lactic  acid  1.  Hydrochloric  acid 

2.  Oxalic  acid  8.  Formic  acid  2.  Phosphoric  acid 

3.  Nitric  acid  9.  Malic  acid  3.  Sulphuric  acid 

4.  Sulphuric  acid  10.  Acetic  acid  4.  Lactic  acid 

5.  Tartaric  acid  11.  Butyric  acid 

6.  Citric  acid  12.  Valeric  acid 

The  relative  actions  of  the  acids  are  also  dependent  on  the 
nature  of  the  digested  protein  (Berg  and  G  r  i  e  s,  Journ. 
of  Biol.  Chem.,  1907,  2,  489). 

Attempts  have  naturally  often  been  made  to  connect  the 
accelerating  actions  of  the  acids  with  their  strengths  (affinity 
constants  or  degrees  of  dissociation).  To  the  complicated  nature 
of  the  reaction  is  due  the  fact  that,  between  these  two  magnitudes 
no  quantitative  relation,  but  at  most  an  approximate  cor- 
respondence, has  been  found.  A  large  part  of  the  acid  must 
form  salts  with  the  digesting  protein  and  it  appears  that  it  is 
just  these  salts  which  are  the  cause  of  peptic  decomposition. 
The  hydrolysis  of  the  protein  salt,  i.e.,  the  hydrochloride,  must 
diminish  and  the  concentration  of  the  salt  for  a  given  quantity 
of  protein  increase  as  the  strength  of  the  acid  present  increases. 
Since  the  hydrolysis  is  governed  by  the  general  condition  of 
equilibrium 

T2C2  X  Y3C3  =  YiCi  X  T4C4 

protein         acid  salt  water 

where 

y2  and  C2  are  the  degree  of  dissociation  and  the  concentration  of  the  protein 

T3  ;:  c3  «         acid 

*     ;      C*  "  "  "  salt 

Y«         A-  "  »  water, 

it  is  at  once  clear  on  what  magnitudes  the  velocity  of  pepsin- 
digestion  would  depend  and  in  what  manner,  if  the  concentration 
of  the  protein  salt  were  the  sole  determining  factor.  But  first 
of  all  account  must  be  taken  of  the  action  of  the  acid  on  the  enzyme 


96  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

molecule,  which  is  thereby  converted  from  the  inactive  (zymogen) 
into  the  active  condition,  the  acid  presumably  remaining  com- 
bined with  the  enzyme  during  the  whole  course  of  the  digestion. 
Especially  striking  is  the  slight  activity  of  sulphuric  acid 
as  shown  in  the  above  series;  this  is  possibly  to  be  attributed 
to  the  harmful  influence  which  Griitzner  (Pfleiderer, 
Pflug.  Arch.,  1897,  66,  605)  found  to  be  exerted  by  sulphates 
even  in  very  small  amounts.  On  the  other  hand,  the  very  strong 
activating  action  of  oxalic  acid  (W  roblewski  and  others) 
must  be  noticed.  The  position  assigned  by  S  j  6  q  u  i  s  t  to 
phosphoric  acid  is  possibly  related  to  the  activating  influence 
often  found  to  be  exerted  by  phosphates. 

Sorensen  has  made  a  thorough  investigation  of  the 
influence  of  the  concentration  of  the  hydrogen  ions  on  the  velocity 
of  digestion;  this  will  be  referred  to  in  Chapter  IV.  It  appears 
that  the  optimal  action  takes  place  in  solutions  the  concentra- 
tion of  which  with  regard  to  hydrogen  ions  is  about  0-06-normal. 
The  concentration  of  the  free  hydrochloric  acid  of  the  gastric 
juice  was  measured  in  1889  by  F.  A.  Hofmann  (Zentralbl.  f. 
klin.  Med.,  1891,  11)  by  the  physico-chemical  inversion  method. 
There  are,  however,  objections  to  the  use  of  this  method  for  such 
measurements  (compare  Sorensen,  Biochem.  Z.,  1909,  21, 
144). 

As  regards  the  quantitative  determination  of  the  influence 
of  acids  and  bases,  it  is  undoubtedly  best  to  investigate  the  rela- 
tion between  the  velocity  of  the  reaction  and  the 
concentration  of  the  hydrogen  ions  in  the 
solution.  As  Sorensen  has  shown,  the  concentration  of  the 
hydrogen  ions  is  most  conveniently  measured  either  electro- 
metrically  or  colorimetrically  by  means  of  indicators. 

Whether  the  activation  of  a  zymogen  by  acids  is  a  process 
differing  from  the  acceleration  of  the  action  of  an  enzyme  already 
in  the  active  state  has  not  yet  been  clearly  established.  In  so 
far  as  the  a  u  t  h  o  r  's  experiments  go,  no  such  difference  exists 
and  in  what  follows  these  two  effects  will  not  be  treated  separately. 
Reynolds  Green  (Proc.  Roy.  Soc.,  1890,  48,  370) 
assumed  that  an  acid  is  necessary  for  the  activation  of  the  lipase- 
zymogen  of  plants,  and  the  subsequent  lipolysis  also  depends  on 
the  presence  of  dilute  acid. 

From  the  results  of  his  experiments  on  the  influence  of  acids 


ACTIVATOKS,   PAKALYSORS  AND  POISONS  97 

on  the  splitting  of  fats  by  Ricinus-lipase,  H  o  y  e  r  (Chem.  Ber., 
1904,  37,  1436)  drew  the  conclusion  that,  for  a  given  amount 
of  fat  or  enzyme  a  definite,  absolute  quantity  of  acid  is  necessary 
for  obtaining  the  optimal  effect.  Sulphuric,  oxalic,  formic, 
acetic  and  butyric  acids  are  about  equal  in  their  capacity  for 
initiating  the  enzyme  action.  Armstrong  and  O  r  m  e  r  o  d, 
whose  experimental  numbers  are  quoted  in  the  next  chapter, 
also  found  no  connection  between  the  activating  actions  of  dif- 
ferent acids  and  their  dissociation  constants.  From  the  fact 
that,  for  a  constant  quantity  of  seeds,  a  definite  minimal  amount 
of  acid  is  required  for  the  maximum  fat-splitting  action,  H  o  y  e  r 
concluded  that  the  acid  reacts  chemically  with  the  seeds  during 
the  decomposition  of  the  fat. 

V  o  1  h  a  r  d  (Zeitschr.  f.  klin.  Med.,  1901,  42,  414  and  43, 
397)  has  carried  out  a  series  of  interesting  experiments  on  the 
sensitiveness  of  gastric  lipase  to  acid  and  alkali.  Gastric  juice 
hydrolyses  fat  in  both  neutral  and  acid  solutions,  a  concentra- 
tion corresponding  with  0-1-normal  hydrochloric  acid  being 
required  to  diminish  the  action  appreciably;  on  the  other  hand, 
the  juice  is  extremely  sensitive  to  minimal  quantities  of  sodium 
hydroxide.  The  glycerine  extract  is,  however,  very 
sensitive  towards  hydrochloric  acid  and  much  more  resistant 
to  alkali.  It  may  hence  be  concluded  that  the  mucous  membrane 
of  the  stomach  contains  a  lipasogen  which,  in  its  behaviour 
towards  acids  and  alkalis,  differs  from  the  stomach  lipase  (the 
so-called  steapsin)  itself. 

Blood-lipase,  investigated  by  R  o  n  a  (Biochem.  Z., 
1911,  33,  413),  exhibits  its  optimal  activity  with  a  H+  — con- 
centration of  1.10~7  —  0-26.10"8,  i.e.,  with  an  approximately 
neutral  reaction. 

According  to  L  i  n  t  n  e  r  and  others,  the  action  of  malt 
diastase  is  accelerated  only  by  excessively  small  quantities 
of  weak  acids.  Ford  finds  that  the  optimal  action  occurs  in 
neutral  solution.  Concentrations  of  acid  as  low  as  0-001% 
of  hydrochloric  acid,  cause  retardation  (Effront,  Cole). 

But  with  pancreas-diastase,  rather  higher  concen- 
trations of  hydrogen  ions — about  0-001-normal — are  necessary 
for  the  activity  to  reach  its  maximum. 

Saliva-amylase  (ptyalin)  was  first  examined 
in  its  relations  to  acidity  and  alkalinity  by  Hammarsten. 


98 


GENERAL  CHEMISTRY  OF  THE   ENZYMES 


Numerous  subsequent  investigations  have  been  made,  with  vary- 
ing results  (Chittenden,  L  a  n  g  1  e  y) .  It  is  active  in 
faintly  alkaline  and  more  so  in  neutral  solution,  while  dialysed 
ptyalin  acts  on  dialysed  starch  still  better  in  an  extremely  faintly 
acid  solution  (Cole,  Journ.  of  Physiol.,  1903,  30,  202,  281). 
But  even  0-001%  of  hydrochloric  acid  exerts  a  retarding  action. 
According  to  F  o  a 's  measurements,  saliva  is  approximately 
neutral. 

Inulinase  also  exhibits  its  optimal  activity  in  a  solution 
having  a  very  slight  acid  reaction  (0-0001-normal  HC1).  Even 
1-5%  soda  solution  completely  destroys  the  enzyme 
(Bourquelot)  . 

Concerning  the  optimal  acidity  of  invertase  we  have  very 
detailed  information.  Apart  from  the  older  work  of  O  '  S  u  1  - 
1  i  v  a  n  and  T  o  m  p  s  o  n  and  of  Cole,  two  important  series 
of  experiments  have  recently  been  made  by  Hudson  and  by 
Sorensen. 

The  following  curve  (Fig.  2)  is  taken  from  H  u  d  s  o  n  's 
work;  it  refers  to  the  temperature  32°  and  gives  the  total 


601 


20 


.002  .004  .006 

Concentration  of  H  Cl 

FlG.  2. 


.008 


.010 


concentration  of  the  hydrochloric  acid  in  solution,  i.e.,  the  sum 
of  the  free  and  combined  (with  traces  of  protein)  acid. 


ACTIVATORS,  PARALYSORS  AND   POISONS 


99 


Hudson  has  proposed  the  following  theory  in  explanation 
of  the  influence  of  acids  and  bases  on  the  activity  of  invertase 
(Journ.  Amer.  Chem.  Soc.,  1910,  32,  1220).  Starting  from  the 
fact  that  invertase  behaves  as  an  amphoteric  electrolyte  and  is 
hence  capable  of  combining  with  both  acids  and  bases,  he  regards 
the  activity  of  the  enzyme  solution  as  proportional  to  the  amount 
of  enzyme  which  is  not  so  combined.  The  values  of  the  activity 
calculated  in  this  way  are  found  to  correspond  with  the  exper- 
imental numbers. 

Sorensen  (Biochem.  Z.,  1909,  21,  144)  found  that  the 
optimal  proportion  of  sulphuric  acid  varies 
widely  for  different  invertase  solutions  and,  as  would  be  expected, 
increases  with  the  nitrogen-content  of  the  enzyme  solution; 
a  similar  relation  would  doubtless  be  found  in  the  case  of  other 
strong  acids.  On  the  other  hand,  the  optimal  concen- 
tration of  the  free  hydrogen  ions  was  found 
to  be  the  same  to  within  0-00003  (p#  =  4-4— 4-6)  in  all  the  series 
of  experiments,  quite  independently  of  the  proportion  of  proteins, 
etc.,  in  the  invertase  solution.  The  position  of  the  optimum 
may  be  seen  from  Fig.  3.  The  three  series  of  experiments  were 
made  with  sulphuric  acid  at  a  temperature  of  52°. 


•8.5',        4.0  4.5  5.0  5.5  6.0  6.5 

Exponent  of  the  Hydrogen-ion  Concentration 

FIG.  3. 

The  concentration  of  the  hydrogen  ions  influences  also  the 
course  of  the  reaction,  i.e.,  the  constancy  of  the  values  of  k 


100 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


calculated   from   the    formula:    k  =  - 


(S  6  r  e  n  s  e  n); 


further  reference  will  be  made  to  this  in  Chapter  IV. 

As  regards  the  influence  of  acidity  on  the  destruction  of 
the  invertase,  E  u  1  e  r  and  .af  U  g  g  1  a  s  showed  that  at  50° 
the  stability  is  a  maximum  when  the  concentration  of  the  hydro- 
gen ions  is  10~6.  At  lower  temperatures  this  optimum  is, 
naturally,  not  so  well  denned,  as  is  shown  by  curves  given  by 
Hudson  and  Paine  (Journ.  Amer.  Chem.  Soc.,  1910,  32, 
779).  The  numbers  given  by  these  investigators  are  as  follows: 


Concentration 
(grm.-mols.  per 
litre)  normal  HC1. 

Rate  of  destruction 
of  invertase 
foX  1000 

Concentration 
(grm.-mols.per 
litre). 

Rate  of  destruction 
of  invertase, 
foX  1000. 

0-05 

365 

Distilled  water 

0 

0-04 

96 

Normal  NaOH 

0-03 

42 

0-01 

3 

0-02 

4 

0-02 

11 

0-015 

1 

0-03 

50 

0-01 

0 

0-04 

245  . 

The  rate  of  destruction,  fo,  was  calculated  from  the  activity  A. 
The  destruction  follows  the  formula  for  unimolecular  reactions, 

1  A 

namely,  —  log  -r- —  =  £2,  where  A  is  the  activity  of  the  invertase 
t          A.  —  x 

at  the  beginning  of  the  destruction  aiid  x  is  the  activity  after 
the  destruction  has  proceeded  for  t  minutes. 

L  a  c  t  a  s  e.  According  to  B  i  e  r  r  y  and  S  a  1  a  z  a  r  (C.R., 
1904,  139,  381)  the  optimal  action  takes  place  with  an  acidity 
of  0-001  N-HC1;  0-01  N-acid  has  a  retarding  action. 

Pectinase  (Bertrand's  pectase)  shows  no  action  at 
all  in  0-1%  hydrochloric  acid  solution. 

Among  the  proteolytic  enzymes  there  are  some — especially 
from  plants — which,  as  already  mentioned,  show  their  optimal 
activity  in  a  slightly  acid  solution.  This  is  the  case,  according 
to  Wei  s  (H.,  1900,  31,  79)  and  Lintner  (Zeitschr.  f.  d. 
gesamt.  Brauwesen,  1902,  25,  365),  with  the  enzyme  of  malt, 
and,  according  to  V  i  n  e  s  (Annals  of  Bot.,  1897,  11,  563;  1898, 
12,  545;  1901,  15,  563;  1902,  16,  1;  1903,  17,  237,  597;  1904, 
18,  289)  with  numerous  plant-extracts  containing  ereptase  and 
with  yeast-extract.  The  autolysis  of  the  substance  of  germinat- 
ing plants  also  proceeds  best  in  acid  solution  (Butkewitsch). 


ACTIVATORS,  PARALYSORS  AND  POISONS  101 

E  ni  u  1  s  i  n  shows  its  optimal  action  in  neutral  solu- 
tion, as  A  u  1  d  has  recently  shown  by  exact  experiments 
(Journ.  Chem.  Soc.,  1908,  93,  1251).  For  the  decomposition  of 
salicin  by  emulsin,  Vulquin  and  Martini*  (Soc.  BioL, 
1911,  70,  763)  give  the  optimal  concentration  of  hydrogen  ions 
asO-36.10~4— 0-41. 10~4. 

P  a  p  a  i  n,  according  to  older  statements,  acts  best  in  neutral 
solution;  it  is  weakened  by  alkali,  the  activity  being  restored 
by  hydrochloric  acid  (H.,  1907,  51,  488). 

Similarly,  the  animal  body  contains,  in  addition  to  peptase, 
proteolytic  enzymes  which  exhibit  their  action  in  acid  solution. 
Among  these  are  the  proteolytic  enzymes  of  the  lymphatic  glands, 
kidneys  and  spleen  which  were  discovered  by  H  e  d  i  n  and 
Rowland  (H.,  1901,  32,  341)  and  are  retarded  by  alkali. 

Numerous  researches  on  the  autolytic  enzymes 
are  in  agreement  in  indicating  that  autolysis  takes  place  only 
in  acid  solution  or,  at  any  rate,  that  it  proceeds  much  more 
rapidly  in  acid  than  in  faintly  alkaline  solution  (S  c  h  w  i  e  n  i  n  g, 
Virch.  Arch.,  1894,  136,  444;  Hildebrandt,  Hofm.  Beitr., 
1904,  5,  463;  von  Drjewezki,  Biochem.  Z.,  1906,  1, 
299) .  A  detailed  investigation  of  the  influence  of  acids  and  alkalis 
on  autolysis  is  due  to  H  e  d  i  n. 

Within  certain  limits  of  concentration,  boric  and  salicylic 
acids  cause  increase  of  the  autolysis  of  the  liver  over  that  occurring 
in  chloroform-water.  With  the  optimal  concentration,  the 
following  proportions  of  the  total  nitrogen  pass  into  solution 
(Y  o  s  h  i  m  o  t  o,  H.,  1909,  58,  341). 

In  chloroform- water 21  •  6% 

In  1%  boric  acid  solution 40-8% 

In  half -saturated  salicylic  acid  solution 47-4% 

Similar  results  were  obtained  by  K  i  k  k  o  j  i  (H.,  1909, 
63,  109). 

According  to  Sachs  (H.,  1905,  46,  337)  the  nucleases  act 
best  in  faintly  acid  solution. 

Pancreatic  and  intestinal  juices,  which  act 
preferably  with  a  neutral  or  alkaline  solution,  are  not  quite 
so  sensitive  to  acids  as  the  gastric  juice  is  to  alkalis.  In  aqueous 
solution  pure  tryptase  is  rendered  inactive  by  a  0-01-JV  con- 
centration of  hydrogen  ions.  But  presence  of  the  substrate 


102  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

diminishes  the  sensitiveness  and  0-02%  of  lactic  acid  even 
accelerates  tryptic  digestion  (Lindberger).  Hence  tryptase 
and  ereptase  hydrolyse  proteins  not  only  in  alkaline  or  neutral 
solutions,  bufc  in  certain  cases  also  in  slightly  acid  solutions. 
But  it  is  found  that  even  free  carbonic  acid  retards  the  digestion. 
With  trypsin,  the  origin  of  the  enzyme  also  causes  irregularities 
in  this  respect;  such  irregularities  have  led  Pollak  (Hofm. 
Beitr.,  1904,  6,  95)  to  assume  the  existence  of  a  separate  enzyme, 
giutinase. 

Just  as  the  optimal  acidity  is  given  by  the  concentration 
of  the  hydrogen  ions  in  solution,  so  the  optimal  alkalinity  is 
given  by  the  concentration  of  the  free  hydroxyl  ions.  From 
the  results  of  D  i  e  t  z  e  (Dissertation,  Leipzig,  1900),  K  a  n  i  t  z 
(H.,  1902,  37,  75)  has  calculated  that  tryptic  digestion  is  most 

rapid  when  the  solution  has   a  concentration normal 

with  respect  to  hydroxyl  ions. 

These  values,  which  were  obtained  for  fibrin-digestion,  cannot, 
however,  be  applied  immediately  to  all  trypsin  actions. 

According  to  Kudo  (Biochem.  Z.,  1909,  15,  473),  tryptase- 
digestion  is  retarded  by  an  alkali-concentration  of  0-0118% 
or  0-003-normal.  The  reversible  retarding  action  is  accompanied 
by  an  irreversible  action,  which  destroys  the  tryptase. 

In  experiments  on  the  velocity  of  decomposition  of  dipeptides 
by  pancreatin  and  by  ereptase,  the  author  (H.,  1907,  57, 
213)  obtained  very  low  results.  Ereptase  from  pig-intestine 
showed  a  marked  alkalinity-optimum,  as  is  seen  from  the  following 
figures  obtained  with  glycylglycine : 

0-1  N-glycylglycine.     5  grms.  powdered  ereptase  per  100  c.c. 

Concentration  of  alkali 0  0-04      0-05      0-075      0-10 

Reaction  constant,  k X 1000 <0-05      7-0        6-2        2-6          0-2 

With  erepsin  from  germinating  peas,  the  optimum  was  not  so 
sharp  (Arkiv  for  Kemi,  1907,  2,  No.  39). 

Concentration  of  alkali 0          0-025      0-05        0-10 

Reaction  constant,  k X 10000 0-7     10-3          9-0          5-7 

When  allowance  is  made  for  the  salt-formation  between  the 
alkali  and  the  glycylglycine,  the  dissociation  constant  of  which 
as  an  acid  amounts  to  1-8X10"8,  the  first  of  these  tables  gives 


ACTIVATORS,   PARALYSORS  AND  POISONS  103 

as  the  optimal  concentration  of  the  hydroxyl  ions,  0  -00002-normal. 
(Combination  of  the  erepsin  with  alkali  is  here  disregarded.) 

Abderhalden  andKoelker  (H.,  1908,  54,  363) 
have  carried  out  similar  experiments,  some  of  the  results  of  which 
(series  B,  p.  380)  are  given  below: 

(a)  1-5  mol.  NaOH      (6)  1-0  mol.  NaOH       (c)  Without  NaOH,  as 

(calculated  on  the  amount  of  dipeptide  taken)  control. 

4-0  c.c.  of  a  ^j  mol.  4-0  c.c.  of  a  ^  mol.  4-0  c.c.  of  a   ^  mol. 

solution  of  glycyl-       solution    of  glycyl-      solution    of    glycyl- 

Z-tyrosine.  Z-tyrosine.  Z-tyrosine. 

0*6    c.c.     pancreatic  0-6     c.c.    pancreatic  0-6     c.c.      pancreatic 

juice.  juice.  juice 

0-07    c.c.    intestinal  0-07    c.c.    intestinal  0-07     c.c.     intestinal 

juice  juice  juice 

0  •  55  c.c.  N-NaOH      0  •  37  c.c.  N-NaOH        1  •  6  c.c.  water 
Time  in    *  "^  C<C'  water  1  "23  c.c.  water 

Minutes.  Rotations. 


6 

+0-80° 

+0-73° 

+0-59° 

15 

+0-81° 

+0-75° 

+0-57° 

41 

+0-80° 

+0-64° 

+0-48° 

174 

+0-76° 

+0-60° 

+0-40° 

260 

+0-73° 

+0-54° 

+0-38° 

378 

+0-58° 

+0-43° 

+0-23° 

1428 

+0-49° 

-J-0-310 

-f-0-090 

These  results  show  that  even  small  quantities  of  alkali  retard 
the  hydrolysis  of  glycyl-Z-tyrosine  by  pancreatic  juice + intestinal 
juice. 

A  number  of  investigations  on  the  influence  of  alkalinity  on  the 
decomposition  of  proteins  and  peptones  by  erepsin  have  been 
carried  out  by  Vernon  (Journ.  of  PhysioL,  1903,  30,  330; 
1904,  32,  33).  He  distinguishes  two  ereptases — pancreatic  and 
intestinal — both  of  which  are  accelerated  by  0  •  4-1  •  2%  of  sodium 
carbonate,  the  intestinal  ereptase  being  at  the  same  time  irre- 
versibly destroyed.  The  erepsins  of  different  animals  exhibit 
varying  sensitiveness  towards  alkali;  the  protective  action  of 
the  proteins,  studied  by  V  e  r  n  o  n  (cf.  p.  115)  may  here  be  the 
determining  factor. 

Also  the  investigations  of  B  a  y  1  i  s  s  (Arch.  Sci.  Biol.  St. 
Petersburg,  1904,  11,  261,  Supplement)  on  casein  indicate  a 
smaller  optimal  alkali-concentration  than  D  i  e  t  z  e  's  •  exper- 
iments with  fibrin. 

We  have  as  yet  no  clear  conception  concerning  the  mode 
of  action  of  the  alkali  in  tryptic  digestion.  During  the  reaction 


104  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

free  alkali  must  disappear,  since  carboxyl  groups  are  rendered 
free  by  the  division  of  the  protein-molecule  into  amino-acids. 
It  might  then  be  expected  that  this  diminution  of  the  hydroxyl 
ions  would  be  rendered  apparent  in  the  course  of  the  reaction. 
The  obvious  step  is  to  test  the  applicability  of  one  of  the  formulae 
holding  for  negative  auto-catalyses — processes  in  which  the 
catalyst  is  used  up  by  the  reaction  itself.  It  is  found,  however, 
that  such  formulae  do  not  correspond  with  the  experimental 
numbers;  on  the  contrary,  in  the  experiments  of  Taylor 
and  B  a  y  1  i  s  s,  the  coefficient  k  of  the  theoretical  formula 

1  7      a 

k  —  —  ln 


t      a—x 

remains  comparatively  constant.  With  the  help  of  a  gas  chain, 
T.B.Robertson  and  Schmidt  (Journ.  of  Biol.  Chem., 
1908,  5,  31)  have  recently  studied  the  law  according  to  which  the 
hydroxyl  ions  diminish  in  concentration  during  the  digestion. 
They  find  that  this  diminution  may  be  expressed  by  a  unimolec- 
ular  formula  if  the  OH-concentration  is  greater  than  10  ~6  and 
by  a  bimolecular  formula  if  this  concentration  lies  between  10  ~6 
and  10  ~7.  Definite  conclusions  in  regard  to  the  part  played  by 
the  OH-ions  in  the  digestion  cannot  be  drawn  from  these  results. 

Hexosephosphatese,  the  enzyme  that  effects  the 
esterification  of  the  hexoses  with  phosphoric  acid  acts  only  in 
neutral  or  alkaline  solution  (E  u  1  e  r  and  Kullberg,  H.,  1911, 
74,  15). 

Chymase  (chymosin)  is  converted  from  the  state 
of  zymogen  into  the  active  condition  by  acids  (  H  a  m  m  a  r  - 
s  t  e  n,  1872)  which,  according  to  their  efficiency  in  this  respect, 
are  arranged  in  the  following  order  (equimolecular  proportions): 
HC1,  HNO3,  H2S04,  lactic,  acetic,  H3P04  (L  6  r  c  h  e  r,  Pfliig. 
Arch.,  1898,  69,  183).  After  the  rennet  is  activated,  it  functions 
in  either  neutral  or  alkaline  solution.  The  differences  between 
rennets  of  various  origins  have  been  clearly  indicated  by  a  series 
of  investigations  by  G  e  r  b  e  r  (C.  R.,  1907-1910,  145-150; 
see  references  on  p.  48). 

The  action  of  vegetable  rennases,  which  at  all  temperatures  clot 
raw  milk  less  easily  than  boiled,  is  retarded  by  small  quantities  of 
alkali  and  accelerated  by  larger  quantities  of  acid.  Those  which  act 
on  fresh  milk  with  difficulty  only  at  high  temperatures  are  retarded  by 


ACTIVATORS,  PARALYSORS  AND  POISONS  105 

acids  having  a  higher  basicity  than  two  and  also  by  quite  small  pro- 
portions of  dibasic  acids,  larger  proportions  of  which  have  an  accelerating 
action;  all  other  acids  exert  an  intensifying  effect.  But  those  chymosins 
which  clot  raw  milk  more  readily  than  boiled,  are  accelerated  in  their 
action  by  all  acids. 

With  certain  (calciphile)  vegetable  rennets,  e.g.,  that  from  the  sap 
of  Madura  aurantiaca,  the  clotting  of  both  raw  and  boiled 
milk  is  accelerated.  In  presence  of  basiphile  rennet,  the  clotting  of 
raw  milk  is  only  slightly  hastened  by  small  doses  of  HC1  and  is  con- 
siderably retarded  by  medium  amounts  of  the  acid;  the  action  on  boiled 
milk  is  in  all  cases  accelerated,  but  to  a  much  less  extent  than  with  the 
really  calciphile  rennases,  such  as  chymosin  from  calf's  stomach. 

The  concentration  of  the  H'-ions  in  milk  has  recently  been  measured 
electrometrically  by  van  Dam  (H.,  1908,  58,  295)  and  found  to  be 
0  •  14-0  •  32  X  10~6 ;  the  results  obtained  indicate  that  the  time  of  clotting 
is  inversely  proportional  to  this  concentration. 

Parachymase  (parachymosin)  seems  to  be  much 
more  resistant  to  acids  but  much  more  easily  destroyed  by  alkali, 
than  chymosin  (Bang,  Pfliig.  Arch.,  1900,  79,  425). 

Sera  showing  slight  t  h  r  o  in  b  i  n  action  are  activated  by 
either  acids  or  alkalis  (Arch.  f.  klin.  Med.,  79  and  80). 

Z  y  m  a  s  e-f  er  mentation  is  accelerated  by  small  quan- 
tities of  alkali  (B  u  c  h  n  e  r  and  R  a  p  p  ,  Chem.  Ber.,  1897, 
30,  2668)  and  retarded  by  slight  amounts  of  acid. 

The  remarkable  sensitiveness  of  1  a  c  c  a  s  e  to  acid,  which 
was  determined  quantitatively  by  Bertrand,  is  shown 
by  the  numbers  given  in  the  next  chapter. 

Peroxydases  are  also  paralysed  by  acids,  of  which 
larger  quantities  are  required  than  in  the  case  of  laccase.  The 
paralysing  effect  of  acids  is  approximately  proportional  to  their 
degree  of  dissociation  (Bertrand  and  Rozenband, 
C.  R.,  1909,  148,  297). 

The  influence  of  acids  and  alkalis  on  blood-catalases  of  various 
origins  was  first  studied  in  detail  byJacobson  (H.,  1892, 
16,  340).  The  author  has  compared  the  behaviour  of  the 
catalases  from  fat  and  from  Boletus  s  c  a  b  e  r  (Hofm. 
Beitr.,  1905,  7,  1). 

Senter  (Zeitschr.  f.  physikal.  Chem.,  1903,  44,  257) 
found  that  acids  cause  considerable  retardation  of  the  action  of 
catalase,  without  injuring  the  enzyme  permanently.  The  length 
of  the  incubation  period — the  time  during  which  the  enzyme  is 


106  GENERAL  CHEMISTRY  OF  THE   ENZYMES 

in  contact  with  the  acid  before  the  hydrogen  peroxide  is  added — 
has  no  substantial  influence  on  the  reaction.  That  very  low 
concentrations  of  acid  have  a  large  effect  is  shown  by  the  following 
figures : 

Concentration  of  acid  Velocity  constant 

1/10,000-normal  HC1 0-0011 

1/20,000-normal  HC1 0-0075 

I/ oo -normal  HC1 0-0100 

With  hydrochloric  and  nitric  acids  the  retarding  action, 
according  to  S  e  n  t  e  r  ,  varies  very  nearly  proportionally  with  the 
third  power  of  the  acid-concentration.  But  Faitelowitz 
states  that  the  "  poisoning  "  of  milk-cat alase  by  hydrochloric 
acid  is  approximately  proportional  to  the  concentration  of  the 
acid. 

In  connection  with  the  action  of  acids  and  bases,  mention 
must  be  made  of  that  of  acid  salts,  of  which  the  acid  phos- 
phates, carbonates,  citrates,  etc.,  have  more  especially  to  be  con- 
sidered. In  this  case,  also,  the  hydrogen  ions  usually  constitute 
the  active  component.  Acid  salts,  or  mixtures  of  neutral  salts 
with  the  corresponding  (weak)  acids,  maintain  the  concentration 
of  the  hydrogen  ions  in  enzyme  reactions  constant  within  fairly 
narrow  limits,  since  in  their  presence  small  quantities  of  acids 
or  bases  can  only  cause  a  slight  change  in  the  ionic  equilibrium 
of  the  solution.  Such  acid  salts  or  mixtures  of  salts  are  appro- 
priately termed  "  buffers."  As  will  be  readily  understood, 
amino-acids  act  in  the  same  manner  as  acid  salts,  i.e.,  as  buffers. 

Activation  by  Salts.  As  has  been  pointed  out  by 
Delezenne  and  E.  Z  u  n  z  (Soc.  Biol.,  1906,  59,  477  and  60, 
1070),  calcium  and  magnesium  salts  exert  a  very  marked  activat- 
ing and  accelerating  influence  on  tryptic  digestion .  [The  harmful 
effect  of  calcium  chloride,  found  by  Malfitano  (C.R., 
1905,  141,  912)  with  the  protease  of  splenic  fever  is  perhaps  due 
to  the  optinmm  concentration  of  calcium  being  exceeded.] 

It  is  assumed  by  Pawl  ow,  by  Bayliss  and  Star- 
ling (Journ.  of  Physiol.,  1905,  32,  129),  and  by  Zunz  that 
the  transformation  of  pro-enzymes  by  kinases  or  by  calcium  salts 
represents  a  catalytic  reaction.  In  support  of  this  view  E  . 
Zunz  has  made  the  following  experiments : 


ACTIVATORS,   PARALYSORS  AND  POISONS  107 

After  inactive  pancreatic  juice  had  been  left  for  10-12  hours 
in  an  incubator  with  calcium  chloride  or  nitrate  and  the  calcium 
precipitated  by  ammonium  oxalate,  it  was  found  to  have  a  pro- 
teolytic  action.  Under  the  same  conditions  the  juice  was  not 
activated  in  1-2  hours.  Analogous  results  were  obtained  with 
magnesium  salts. 

When  added  in  the  form  of  soluble  salts,  sodium,  potassium, 
ammonium,  zinc,  beryllium,  aluminium,  cobalt,  nickel,  iron, 
manganese,  uranium  and  copper  had  no  effect.  With  salts  of 
caesium,  rubidium  and  lithium,  an  activating  influence  was  shown, 
but  not  regularly.  For  the  numerous  detailed  results  obtained 
by  Z  u  n  z  with  regard  to  the  activation  of  pancreatic  juice, 
reference  should  be  made  to  his  complete  monograph  "  Recherches 
sur  Tactivation  du  sue  pancreatique "  (Brussels,  1906-1907). 

a-Pro-thrombase  is  converted  by  calcium  salts  into  thrombase 
(M  o  r  a  w  i  t  z,  Hofm.  Beitr.,  1903,  4,  381;  1904,  5,  133).  Also 
pectinase,  the  vegetable  clotting  enzyme  which  transforms 
pectin  into  pectinic  acid,  functions  only  in  presence  of  calcium 
(or  barium  or  strontium)  salts  (Bertrand  and  M  a  1 1  e  v  r  e , 
C.R.,  1894,  119,  1012;  1895,  120,  110  and  121,  726). 

As,  in  addition  to  tryptase,  ereptase  (A  b  d  e  i  h  a  1  d  e  n, 
Caemmerer  and  Pinkussohn,  H.,  1909,  59,  293)  and 
pancreatic  lipase  are  intensified  in  their  action  by  calcium  chloride 
(Pottevin,  C.  R.,  1903,  136,  767;  Kanitz,  H.,  1905,  46, 
482),  it  can  no  longer  be  asserted  that  calcium  is  a  strictly  specific 
activator.  There  is,  however,  no  doubt  that,  in  the  cases  named, 
calcium  cannot  be  replaced  by  the  alkali  metals. 

That  the  well-known  discoveries  of  Jacques  Loeb 
(Vorlesungen  liber  die  Dynamik  der  Lebenserscheinungen,  Leipzig, 
1906)  on  the  specific  action  of  calcium  salts  are  related  to  these 
phenomena  can  scarcely  be  doubted. 

On  the  other  hand,  purely  chemical  reactions  are  also  known 
in  which  calcium  acts  as  a  specific  catalyst.  Thus,  O  .  L  o  e  w 
(Chem.  Ber.,  1888,  21,  270)  found  that  lime  is  an  especially 
suitable  agent  for  the  condensation  of  formaldehyde  to  sugar. 
The  author  has  observed  the  same  to  be  the  case  with  the 
formation  of  formate  (Chem.  Ber.,  1905,  38,  2551). 

A  specific  action,  similar  to  that  of  calcium,  appears  to  be 
exerted  in  certain  cases  by  magnesium.  The  important 
part  played  by  magnesium  in  plant  life  has  been  referred  to  by 


108  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Willstatter  (Lieb.  Ann.,  1906,  350,  48)  in  a  very  interest- 
ing paper. 

Among  the  metals  of  biological  importance  as  activators, 
manganese  must  also  be  numbered.  As  was  first  established 
by  Bertrand,  this  metal  is  an  essential  constituent  of  the 
oxydases.  It  occurs  in  company  with  hydroxy-acids,  with 
which,  in  the  oxydases,  it  is  combined.  As  manganese  is  known 
to  be  a  powerful  oxidising  catalyst,  its  function  in  the  oxydases 
is  readily  explained  from  a  chemical  standpoint. 

The  accelerating  action  of  manganese  on  the  decomposition 
of  hydrogen  peroxide  by  catalases  is  also  related  to  these 
effects. 

More  remarkable  is  the  fact  that  manganese  salts  hasten 
enzymic  hydrolyses.  Thus,  as  H  o  y  e  r  (Chem.  ZentralbL, 
1905,  II,  582)  found,  the  splitting  of  fat  by  vegetable  Upases  is 
favoured  by  small  quantities  of  manganese  sulphate,  whilst  the 
diastatic  enzymes  of  serum  and  of  pancreatic  juice  were  shown 
by  A .  G  i  g  o  n  and  T. Rosenberg  (Skand.  Arch,  f .  Physiol., 
1908,  20,  423)  to  be  activated  energetically  by  the  same  salt, 
even  in  the  concentration  0-001%. 

According  to  K  a  y  s  e  r  and  Marchand  (C.  R.,  1907, 
144,  574,  714;  1907,  145,  343;  1910,  151,  816)  and  also  Fern- 
bach  and  Lanzenberg,  glucose  is  fermented  more 
quickly  and  completely  in  presence  of  manganese  nitrate  (0  •  1- 
0  •  5%)  than  without  it. 

Iron  salts  serve  as  general  catalysts  for  purely  chemical 
oxidations,  and,  just  as  they  accelerate  the  decomposition  of 
hydrogen  peroxide  by  colloidal  platinum,  they  intensify  the  action 
of  the  catalases,  when  used  either  alone  or  together  with  man- 
ganese salts.  They  also  exert  a  specific  accelerating  influence 
on  the  action  of  the  tyrosinases  (Durham,  Proc.  Roy.  Soc., 
1904,  74,  310;  Bach,  Chem.  Ber.,  1910,  43,364).  Whether 
the  undoubtedly  important  role  of  iron  salts  in  the  organism  is 
played  in  conjunction  with  the  enzymes,  or  whether  it  is  prefer- 
ably independent,  cannot  yet  be  decided. 

According  to  B  a  c  h  (Chem.  Ber.,  1910,  43,  366),  aluminium 
salts  intensify  the  action  of  tyrosinase  even  more  than  manganese 
salts  do.  Less  marked,  but  still  appreciable,  are  the  effects  of 
calcium  and  magnesium  salts. 

It  may  here  be  mentioned  that  hydrogen  peroxide  increases 


ACTIVATORS,  PARALYSORS  AND  POISONS  109 

the  action  of  the  digestive  enzymes  (Vandevelde,  Hofm. 
Beitr.,  1904,  5,  558). 

Among  the  most  noteworthy  of  inorganic  activators  are  the 
alkali  phosphates.  Apart  from  the  action  of  primary 
and  secondary  phosphates  as  "  buffers  ",  the  phosphates  exert 
a  marked,  specific  influence  on  certain  enzyme  reactions. 
Mention  must  first  of  all  be  made  of  zymase-fermentation  for 
which,  as  explained  elsewhere,  the  presence  of  a  phosphate  is 
necessary.  Further,  ammonium  and  calcium  monophosphates 
accelerate  diastatic  action  (E  f  f  r  o  n  t),  whilst  for  the  action  of 
ptyalin,  phosphates  are  absolutely  necessary  (Roger,  Soc. 
Biol.,  1908,  65,  374.)  The  same  is  the  case  with  liver-diastase 
(see  later).  This  effect  possibly  explains  the  following  obser- 
vation made  by  R  o  g  e  r  (Soc.  Biol.,  1907,  62,  833,  1021,  1070) : 

Human  saliva  is  inactivated  by  heating  for  10-15  minutes 
at  85-100°,  but  if  a  small  quantity  of  fresh  saliva  is  subsequently 
added,  the  mixture  has  a  much  greater  saccharifying  action  than 
the  added  saliva  alone.  This  observation  has  recently  been 
extended  and  completed  by  B  a  n  g  (Biochem.  Z.,  1911,  32,  417). 

As  has  been  shown  by  Harden  and  Young,  the  phos- 
phates play  an  extremely  important  part  in  fermentation.  These 
actions  are  described  in  detail  elsewhere  (Chapters  I  and  IV). 

Rennetic  action  is  also  favoured  by  small  quantities  of  mono- 
sodium  phosphate  (G  e  r  b  e  r  ,  Soc.  Biol.,  1908,  64,  1176) 
possibly  owing  to  the  alteration  effected  in  the  acidity.  The  same 
cause  may,  perhaps,  explain  the  activation  of  laccase  by  disodium 
phosphate  in  certain  oxidations  (J.Wolff,  C.  R.,  1909, 
149,  467). 

In  connection  with  these  intensifications  by  phosphoric  acid, 
it  may  be  mentioned  that  Chittenden  observed  an  accel- 
eration of  peptic  digestion  byarsenious  acid.  Accord- 
ing to  I  z  a  r  ,  autolysis  is  sometimes  hastened  by  arsenic. 

Also  in  their  effect  on  alcoholic  fermentation,  arsenates  and 
arsenites  correspond  to  some  extent  with  the  phosphates,  as  is 
shown  by  recent  important  results  obtained  by  Harden  and 
Young  and  referred  to  at  length  in  Chapter  IV. 

Alkali  Salts.  The  salts  of  the  alkalis  are  partly  accel- 
erating and  partly  inhibiting  in  their  action. 

The  salt  which  has  been  most  thoroughly  investigated  is 
sodium  chloride.  According  to  Osborne,  Bierry  and 


L10  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

3  chaffer  (Soc.  Biol.,1907,62,723),  Cole  (Journ.of  Physiol., 
1903,  30,  202,  281),  Wohlgemuth  (Biochem.  Z.,  1908,  9, 
10),  it  facilitates  the  actions  of  diastase,  maltase  and  ptyalin. 
But  the  majority  of  the  enzymes,  e.g.,  invertase,  peptase,  tryptase 
and  the  zymases  and  catalases  (see,  for  instance,  Lockemann, 
T  h  i  e  s  and  W  i  c  h  e  r  n  ,  H.,  1909,  58,  390)  are  retarded  by 
sodium  chloride. 

Amylase  is  accelerated,  often  considerably,  by  small  quantities 
of  the  chlorides,  nitrates,  sulphates,  phosphates,  vanadates  and 
alums  of  the  alkali  metals  (L  i  n  t  n  e  r  ,  Journ.  prakt.  Chem., 
1887,  [2],  36,  481;  E  f  f  r  o  n  t ,  C.  R.,  1892,  115,  1324;  G  r  ii  s  s). 
Ptyalin  is  slightly  accelerated  by  potassium  iodide  (N  e  i  1  s  o  n 
and  Terry,  Amer.  Journ.  of  Physiol.,  1908,  22,  43). 

Cole  came  to  the  conclusion  that  anions  facilitate  and 
cations  weaken  the  action  of  amylase,  the  effects  increasing 
with  the  electro-affinity  (this  magnitude  is  evidently  meant  by 
Cole,  who  uses  the  somewhat  indefinite  expression  "  actinising 
power  ")  of  the  ions. 

The  amylolytic  action  of  pancreatin  is  also  accelerated  by  a 
number  of  salts  (P  r  e  t  i ,  Biochem.  Z.,  1907,  4,  1)  if  these  are 
added  in  dilute  solution. 

Inhibiting  effects  on  amylase  are,  however,  produced  by 
calcium  and  barium  chlorides  and  by  larger  quantities  of  the 
sulphates,  phosphates  and  alums  of  the  alkali  metals. 

According  to  F.  Kriiger,  NaCl,  KC1,  NH4C1,  CaCl2 
and  MgCl2,  in  equivalent  proportions  exert  equal  retarding  effects, 
so  that  it  must  be  concluded  that  the  inhibiting  action  of  the 
anion  predominates.  This  recalls  the  concordant  results  of 
J.  Schiitz  (Hofm.  Beitr.,  1904,  5,  406)  and  Levites 
(H.,  1906,  48,  187)  which  indicate  that  peptic  digestion  is 
retarded  principally  by  the  anions. 

Kudo  found  that  the  digestive  action  of  pancreatin  is, 
in  general,  weakened  by  alkali  salts,  sodium  chloride  producing 
a  rather  greater  effect  than  the  nitrate  or  nitrite;  the  influence 
of  potassium  salts  is  less  than  that  of  sodium  salts. 

Sodium  and  potassium  sulphates  retard  the  rennetic  action 
of  animal  chymosin  in  proportion  to  their  quantity  (if  the  clotting 
effect  is  determined  with  fresh  milk),  whilst  the  coagulating  action 
of  vegetable  rennet  is  increased  by  small  doses,  and  diminished 
by  larger  ones,  of  these  salts. 


ACTIVATORS,  PARALYSORS   AND  POISONS  111 

This  different  behaviour  of  vegetable  and  animal  rennet 
towards  neutral  sulphates  and  towards  Na2HPC>4  and  K2HPO4, 
G  e  r  b  e  r  explains  as  due  to  the  precipitation  of  lime  by  these 
salts,  lime  being  less  necessary  with  the  vegetable  than  with 
the  animal  enzymes.  By  small  quantities  of  acid  sulphates,  such 
as  KHSO4,  both  animal  and  vegetable  rennets  are  accelerated. 

Concerning  the  action  of  activators  in  general,  it  may  be  said 
that: 

As  far  as  the  "  kinase  "  of  tryptase  is  concerned,  this  can 
be  regarded,  on  the  one  hand,  as  a  catalyst  of  the  reaction  tryp- 
sinogen— »trypsin  and,  on  the  other,  as  functioning  like  the  activat- 
ing acids. 

It  is  highly  desirable  that  a  more  extended  series  of  exper- 
iments should  be  made  to  decide  if  the  conversion  of  pro-enzyme 
into  enzyme  is  a  reversible  process. 

There  still  remains  the  possibility  of  a  chemical  reaction 
occurring  between  zymogen  and  kinase  and  in  order  to  obtain 
information  on  this  question,  the  manner  in  which  the  activation 
varies  with  the  time  would  have  to  be  studied  more  closely. 
That  the  process  is  a  relatively  rapid  one  has  been  shown  by 
P  a  w  1  o  w  and  his  collaborators. 

In  one  way  or  another,  the  zymogen  is  often  activated 
"  spontaneously/'  In  most  cases  the  substrate  or  some  foreign 
substance  succeeding  it  yields  the  activator  as  the  result  of  a  slow 
reaction  of  some  kind.  In  an  interesting  investigation  H  o  y  e  r 
(loc.  cit.)  observed  the  appearance  of  lactic  acid  as  such  an 
activating  substance.  If  the  activator  is  a  normal  product  of 
the  substrate  under  the  given  conditions,  the  well-known  case 
of  auto-catalysis  presents  itself.  A  typical  example 
of  such  a  reaction  is  the  spontaneous  decomposition  of  ethyl 
acetate  in  aqueous  solution;  in  this  instance  the  liberated  acetic 
acid  is  the  catalyst  which  accelerates  the  subsequent  hydrolysis 
in  proportion  to  the  concentration  of  its  hydrogen  ions. 

Meanwhile  there  are  no  grounds  for  making  an  essential 
distinction  between  the  activation  of  the  zymogen  with  initiation 
of  the  reaction  and  the  action  of  acids,  alkalis  and  many  salts  in 
accelerating  the  reaction. 

The  action  of  the  latter  substances — often  termed  co-enzymes 
— rests  undoubtedly  on  the  reversible  formation  of  compounds 
of  these  activators,  partly  with  the  substrate  and  partly  with  the 


112  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

enzyme.  In  most  cases  it  is  a  salt-formation  which  takes  place 
(E  u  1  e  r  ,  Hofm.  Beitr.,  1905,  7,  1);  this  occurs  instantaneously, 
so  that  the  "  incubation  periods  "  of  the  accelerating  alkalis, 
e.g.,  with  catalase,  or  those  of  the  inhibiting  acids  are  without 
influence — of  course,  only  so  long  as  disturbing  secondary  reactions, 
decomposing  chemically  the  enzyme  or  substrate,  are  avoided. 

In  many  cases  the  minimal  concentration  determining  the 
optimum  of  acid-  or  alkali-action  must  correspond  exactly  with 
the  quantity  of  acid  or  alkali  necessary  for  the  neutralisation 
of  the  solution  (cf.  Cole,  Journ.  of  PhysioL,  1903,  30,  202). 

The  sensitiveness  of  the  enzymes  towards  acids  and  alkalis 
is,  indeed,  very  great  but  is  quite  conceivable  if  the  very  small 
concentrations  in  which  the  enzymes  themselves  are  present  in 
solution  are  considered.  More  or  less  complete  analogies  are 
found  with  many  well-known  catalytic  processes.  Thus,  hydro- 
quinone  in  a  solution  containing  0-001  normal-manganese  sul- 
phate undergoes  oxidation  very  slowly;  but  if  sufficient  alkali 
is  added  to  combine  with  the  majority  of  the  sulphuric  acid  and 
thus  to  liberate  manganese  hydroxide,  the  oxidation  is  enormously 
accelerated.  The  manganese  sulphate  corresponds  with  the 
enzyme  in  its  inactive  state,  the  alkali  with  the  activator.  In 
hydrolytic  changes,  e.g.,  the  enzymic  inversion  of  cane-sugar, 
the  chemical  reaction  presumably  consists  in  the  activating  acid 
liberating  the  enzyme — which  in  neutral  solution  is  present  as  a 
salt — and  so  bringing  it  into  the  active  condition  (cf.  E  u  1  e  r 
and  B.  af  U  g  g  1  a  s  ,  H.,  1910,  65,  124). 

As  well  as  to  salt-formation,  an  important  part  in  the  activa- 
tion of  enzymes  must  be  attributed  to  the  formation  of  com- 
plex compounds.  It  is  in  this  way  that  the  specific  properties  of 
phosphoric  acid  and  of  calcium  and  manganese  are  exerted, 
as  described  above.  With  manganese  the  capacity  to  form  com- 
plex compounds  with  hydroxylic  bodies  has  been  long  known, 
and  with  calcium,  physico-chemical  investigations  have  rendered 
necessary  the  assumption  that  it  also  yields  such  complexes. 
The  reactivity  of  phosphoric  acid  with  polyhydric  alcohols  has 
often  been  studied  qualitatively,  and  quantitative  experiments 
would  be  of  biological  interest. 

In  the  description  of  pepsin  action  on  p.  95,  it  was  mentioned 
that  hydrochloric  acid  not  only  acts  on  the  enzyme  but  also 
accelerates  the  digestion  by  forming  salts  with  the  proteins. 


ACTIVATORS,  PARALYSORS  AND  POISONS  113 

Similarly,  it  is  doubtless  the  alkali  salts  of  peptones  and 
peptides  which  constitute  the  active  molecules  of  tryptic  diges- 
tion. This  complete  alteration  of  the  reactivity  of  a  substance 
by  salt-formation  has  many  chemical  analogies:  nitrous  acid 
diazotises,  whilst  nitrites  do  not  ;  in  alkaline  solution  polyphenols 
are  oxidised  by  the  oxygen  of  the  air  with  great  readiness,  but 
in  acid  solution  only  with  difficulty.  The  velocity  of  decomposi- 
tion of  hydrogen  peroxide  is  influenced  by  the  acids  or  alkalis 
present  in  a  similar  manner,  no  matter  whether  the  reaction  is 
brought  about  by  "  catalases  "  or  by  inorganic  oxides  (colloidal 
platinum,  ferric  hydroxide). 

A  similar  argument  can  be  applied  also  in  other  cases,  e.g., 
that  of  invertase-action,  where  it  may  be  assumed  that  the 
compound  (cane  sugar-mineral  acid),  which  represents  the  active 
molecule  in  the  process  of  inversion,  is  resolved  catalytically  by 
the  enzyme  into  glucose,  fructose  and  free  acid. 

Being  amphoteric  electrolytes  and  hence  capable  of  forming 
salts  with  either  'acids  or  alkalis,  the  proteins,  peptones  and 
amino-acids  often  exert  indirectly  an  accelerating,  though  seldom 
a  powerful,  action;  as  the  author  has  already  emphasised 
(Ergeb.  der  Physiol.;  1907,  6),  they  regulate' the  concentration 
of  the  free  acid  or  base.1 

Finally,  those  cases  must  be  considered  where  the  activator 
represents  the  common  solvent,  that  is,  the  con- 
necting link  between  enzyme  and  substrate,  where  the 
two  alone  cannot  form  a  homogeneous  system.  The  activation 
of  the  Upases  by  the  bile  acids  is  possibly  to  be  explained 
in  this  way.  The  purely  chemical  (not  enzymic)  splitting  of 
fats  by  naphthalene-stearosulphonic  acid  has  thus  been  inter- 
preted by  T  w  i  t  c  h  e  1 . 

The  causes  underlying  the  actions  of  neutral  salts 
are  also  very  varied. 

These  actions  are  partly  due  to  simple  chemical  transposi- 
tions between  the  electrolytes  present  and  the  consequent  altera- 
tion of  the  acidity  or  alkalinity.  So  that  sodium  sulphate, 
salicylate  and  phosphate  hinder  peptic  digestion  (P  a  w  1  o  w, 
Danilewski),  the  strong  hydrochloric  acid  being  replaced 

1  In  many  instances  the  addition  of  proteins  to  an  enzyme  solution  miti- 
gates the  harmful  action  of  tryptic  ferments  on  the  enzyme.  Beneficial 
effects  of  another  type  are  also  observed,  e.g.,  with  diastase,  ptyalin,  etc. 


114  GENEKAL  CHEMISTRY  OF  THE  ENZYMES 

by  the  weaker,  less  active  acid  of  the  salt.  With  higher  con- 
centrations of  the  salts,  the  influence  of  dissociation  comes  into 
play,  sodium  chloride  diminishing  the  electrolytic  dissociation 
of  the  protein  hydrochloride.  Further  those  influences  come 
into  action  which  produce  the  "  neutral  salt  action  "  in  non- 
enzymic  hydrolyses  and  which  vary  widely  in  different  reactions. 

The  actions  of  small  quantities  of  neutral  salts,  such  as 
NaCl,  KC1,  etc.,  would  indeed  seldom  be  observable  if  the  salt 
were  added  to  enzyme  solutions  previously  free 
from  electrolytes.  Many  investigations  indicate  that 
the  small  quantities,  of  salts  occurring  in  the  organs  with  the 
enzymes  are  essential  for  the  activity  of  the  latter;  if  these  small 
amounts  of  salts  are  removed  by  dialysis,  the  enzyme  action, 
for  example,  of  amylase,  ceases. 

Very  large  quantities  of  salt  have  a  coagulating  action  and 
precipitate  enzymes  more  or  less  directly  from  enzyme  solutions, 
which  usually  contain  colloidal  substances.  Yet  even  quite 
considerable  concentrations  of  salts  are  often  without  harmful 
effect;  thus  the  activity  of  ptyalin  solutions  is  not  weakened  by 
large  proportions  of  magnesium  and  ammonium  sulphates 1 
(Patten  and  Stiles,  Amer.  Journ.  of  Physiol.,  1906, 
17,  26). 

In  addition  to  the  reversible  influence  on  the  time- 
course  of  enzyme  reactions,  acids,  alkalis  and  salts  exert  also  an 
influence  on  the  irreversible  changes  of  the  enzyme- 
substance.  These  two  actions  are  essentially  different.  The 
latter  of  the  two  has  been  studied  in  the  case  of  invertin  by 
Fernbach  (Recherches  sur  la  sucrase,  Thesis,  Paris,  1890). 
This  investigator  found  that  dissolved  invertin,  exposed  to  the 
oxygen  of  the  air,  gradually  undergoes  oxidation  and  becomes 
permanently  inactive;  this  oxidation  occurs  far  more  rapidly 
in  alkaline  than  in  acid  solution.  (Cf .  also  Hudson  and 
Paine,  Journ.  Amer.  Chem.  Soc.,  1910,  32,  774). 

1  It  is  regarded  as  unnecessary  to  mention  the  many  cases  in  which 
smaller  proportions  of  salts  have  no  marked  effect, 


ACTIVATORS,  PARALYSORS  AND  POISONS  115 


PROTECTIVE  AGENTS 

Lastly,  the  influence  of  neutral  salts  on  enzyme  reactions 
makes  itself  felt  in  an  indirect  way,  a  beneficial  effect  being  pro- 
duced by  the  destruction,  alteration  or  removal  of  substances 
which  may  be  classed  together  as  inhibiting  agents. 

These  causes  play  a  part  in  the  interesting  biological 
phenomena  accompanying  fertilisation  described  some  years 
ago  by  Jacques  Loeb. 

The  favourable  influence  of  certain  other  substances  on 
enzyme  solutions  is  also  often  due  to  the  counteraction 
of  the  inhibiting  action  of  the  paralysors. 
In  this  way  we  may  regard  the  acid  salts — often  termed  "buffers  " 
— and  the  proteins  too  as  protective  agents.  The  latter  may, 
for  instance,  unite  with  part  of  the  alkali  in  the  case  of  tryptic 
digestion  and  thus  protect  the  enzyme,  especially  at  relatively 
high  temperatures,  from  destruction  (Vernon,  Journ.  ofPhysiol., 
1904,  31,  346).  Also  soda,  calcium  carbonate,  etc.,  which  are 
able  to  protect  yeast  from  the  poisonous  effect  of  various  substances, 
do  so  in  virtue  of  their  neutralising  action  (Henneberg, 
Centralbl.  f.  Bakt.,  1908,  II,  20,  225). 

INHIBITING  AGENTS   (PARALYSORS) 

The  term  "  poison,"  which  has  recently  been  largely  used  for 
all  substances  which  delay  or  prevent  catalytic  reactions,  is  not 
justifiable  in  the  light  of  recent  knowledge.  The  expression 
poisoning  must  therefore  be  reserved  for  the  disturbance  of  the 
life  functions  by  paralysors. 

Concerning  the  chemistry  of  the  action  of  paralysors  we  are 
almost  completely  in  the  dark,  but  these  bodies  are  of  con- 
siderable interest  in  enzymology  since  they  are  indispensable 
as  sterilising  agents  in  all  protracted  experiments.  The  influence 
which  paralysors,  such  as  chloroform  etc.,  may  at  times  exert  on 
enzymic  decompositions  is  shown,  for  example,  by  E.  F  i  s  c  h  e  r's 
observations  on  the  hydrolysis  of  glucosides  by  yeast-enzymes 
(Chem.  Ber.,  1895,  28,  1436). 

Just  as  with  the  action  of  activators,  that  of  poisons  and  other 
inhibiting  substances  is  dependent  on  the  concentration  of  the 
enzyme  and  the  purity  of  the  solution.  It  is  found  that  the 


116  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

injurious  action  of  poisons  on  enzyme  solutions  increases  as  the 
concentration  of  the  enzymes  diminishes;  this  behaviour  seems 
to  indicate  that  an  addition  of  the  poison  to  the  enzyme  takes 
place. 

Inorganic      Salts 

NaF.  According  to  A  r  t  h  u  s  and  H  u  b  e  r  (C.  R.,  1892, 
115,  839)  this  salt  is  without  influence  on  the  soluble  enzymes, 
but  kills  bacteria.  Its  effect  on  lipase  is  greater  than  that  of 
any  other  antiseptic  (Loevenhart,  Journ.  of  Biol.  Chem., 
1907,  2,  391 ;  K  a  s  1 1  e  and  Loevenhart,  Amer.  Chem. 
Journ.,  1900,  24,  491).  Chymosin  is  injured  by  it,  but  not 
stronger  solutions  of  trypsin  (Kaufmann,  H.,  1903,  39, 
434).  The  action  of  erepsin  on  dipeptides  is  partly  retarded, 
partly  accelerated  by  sodium  fluoride  (Abderhalden, 
Caemmerer  and  Pinkussohn,  H.,  1909,59,  293). 
According  toVandevelde  it  has  no  effect  on  pepsin  and 
trypsin.  B  u  c  h  n  e  r  states  that  ammonium  fluoride  annuls 
the  action  of  zymase.  The  influence  of  fluorides  on  tjirombin 
has  been  thoroughly  investigated  by  B  o  r  d  e  t  and  G  e  n  g  o  u 
(Ann.  Inst.  Pasteur,  1904,  18,  98)  and  that  on  diastases  by 
Ef front . 

HgCl2i  even  in  0  •  00005N-solution  has  a  harmful  effect  on 
catalase.  In  0-001%  solution,  it  is  poisonous  to  amylase  and 
still  more  so  to  urease.  It  injures  ptyalin  or  trypsin  in  0-005% 
solution,  but  its  action  on  erepsin  is  much  less  marked  (E  u  1  e  r  , 
Arkiv  for  Kemi,  1907,  2,  No.  39).  Invertin  is  also  weakened  by 
it,  but  to  a  relatively  small  extent  (D  u  c  1  a  u  x) . 

Hg(CN)2:  exerts  a  decided  inhibiting  action,  although  less 
than  that  of  HgCl2,  on  catalase  (F  a  i  t  e  1  o  w  i  t  z). 

B(OH)s.  D  u  c  1  a  u  x  found  that  this  retards  the  action  of 
chymosin,  but  the  more  recent  results  of  A  g  u  1  h  o  n  (C.  R., 
1909,  148,  1340)  indicate  an  accelerating  effect.  According  to 
the  latter  author,  the  enzymes  which  hydrolyse  carbohydrates, 
glucosides  and  proteins  act,  without  alteration,  in  cold  saturated 
boric  acid  solution;  catalase  is  somewhat  retarded. 

As2Os.  As  Buchner  has  shown  by  an  extended  series 
of  experiments,  arsenic  is  i  n  j  u  r  i  o  u  s  to  cell-free  fermentation, 
but  proteins  and  sugar  act  as  protecting  agents  against  this 


ACTIVATORS,  PARALYSORS  AND  POISONS  117 

poison.  Amylase  (K  j  e  1  d  a  h  1)  and  pepsin  (Asher) 
are  harmfully  affected  by  arsenic  salts. 

EbS:  has  an  injurious  influence  on  catalase  but  is  without 
action  on  pepsin,  trypsin,  diastase  and  emulsin  (Fermi  and 
P  e  r  n  o  s  s  i ,  Zeitschr.  f.  Hygiene,  1894,  18,  83). 

Os  (ozone).  Whilst  hydrogen  peroxide  accelerates  many 
enzyme  actions  and,  so  far  as  is  known,  has  a  slow  destructive 
action  only  on  catalase,  ozone  has  a  harmful  influence,  as  has 
been  shown  by  S  i  g  m  u  n  d  for  most  of  the  more  important 
enzymes,  by  K  a  s  1 1  e  especially  for  lipase  (Journ.  Chem. 
Soc.,  Abs.,  1906,  i,  615)  and  by  Buchner  and  H  o  f  m  a  n  n 
for  zymase  (Biochem.  Z.,  1907,  4,  215). 

H202:  according  to  Vandevelde  (Hofm.  Beitr.,  1904, 
5,  558),  most  enzymes  are  beneficially  affected,  only  catalase 
being  retarded. 

Of  salts  which  have  a  more  specific  injurious  action,  mention 
may  be  made  of  the  following: 

Alkali  sulphates  retard  peptic'  digestion,  as  was  found  by 
Grutzner  (Pfleiderer,  Pfliig.  Arch.,  1897,  66,  605) 
(with  trypsin  there  is  either  slight  retardation  or  no  effect  at  all) . 

CaCl2  has  an  especially  marked  weakening  action  on  invertin 
(D  u  c  1  a  u  x) . 

Iron  salts  are  injurious  to  pepsin  (Asher). 

Potassium  permanganate  strongly  inhibits  lipase  (K  a  s  1 1  e 
and  Loevenhart). 

Nitrates  and  chlorates  are  stated  to  be  intense  catalase- 
poisons. 

For  certain  other  substances  which  hinder  the  action  of 
blood-catalase,  S  e  n  t  e  r  (Zeitschr.  f.  physikal.  Chem.,  1905, 
51,  673)  gives  the  following  concentrations  as  necessary  to  diminish 
the  velocity  of  reaction  to  one-half  its  original  value: 

Paralysor  Grm.-mol.  per  litre 
I2  in  KI  1/50,000 

Hydroxylamine  hydrochloride  1/80,000 

KNO3  1/40,000 

KC1O3  1/40,000 

For  the  catalase  from  frog's  muscle,  C.  G.  Santesson 
(Skand.  Arch.  f.  Physiol.,  1909,  23,  99)  has  recently  obtained 
similar  results. 


118  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Inorganic  colloids  (gold,  platinum,  silver,  arsenic,  copper, 
mercury,  bismuth)  do  not  accelerate  pepsin,  but  in  high  concen- 
trations rather  inhibit  it  (Pinkussohn,  Biochem.  Z.,  1908, 
8,  387). 


Organic   Poisons   and  Inhibiting   Agents 

Chloroform.  The  original  statement  made  by  M  ii  n  t  z  , 
that  chloroform  injures  only  the  micro-organisms  but  not  the 
enzymes,  has  had  to  be  considerably  modified  in  recent  times. 

Chloroform  injures  maltase  (according  to  L  i  n  t  n  e  r  and 
Krober),  amylase,  ptyalin,  yeast-glucase,  pepsin,  rennin  and 
urease,  but  has  no,  or  but  slight  action  on  trypsin,  erepsin,  invertin 
(Fischer),  Aspergillus-maltase  (H  e  r  i  s  s  e  y)  or  zymase. 
For  the  literature  on  this  subject  see  K  a  u  f  m  a  n  n  (H.,  1903, 
39,  434). 

Chloral  completely  destroys  oxydase  (from  L  e  p  i  o  t  a 
americana)  (Kastle  and  Loevenhart,  Chem. 
Zentralbl.,  1906,  77,  i,  1554)  but  injures  myrosin  only  slightly. 

Formaldehyde.  In  40%  concentration,  this  does 
not  destroy  Lepiota-oxydase  (Kastle  and  Loevenhart, 
1  o  c  .  c  i  t .)  but  it  has  an  injurious  effect  on  chymosin  and  amy- 
lase. In  1%  solution,  it  is  without  action  on  erepsin  (E  u  1  e  r  , 
loc.  cit.).  Zymase  is  harmfully  influenced  by  formaldehyde  but 
pepsin  only  by  concentrated  solutions. 

G  1  y  c  e  r  o  1  has  an  appreciable  inhibiting  influence  on 
rennet-action  (R  e  i  c  h  e  1  and  S  p  i  r  o  ,  Hofm.  Beitr.,  1905, 
7,  485). 

Toluene.  This  hydrocarbon,  which  E.  Fischer 
introduced  for  the  sterilisation  of  enzyme  solutions,  is  harmless 
in  the  great  majority  of  cases,  but  urease  is  weakened  by  it. 

Phenol:  exerts  a  deleterious  action  on  pepsin,  amylase  and 
catalase. 

Cresols:  harmless  for  liver-butyrase  (Kastle,  Chem. 
Zentralbl.,  1906,  77,  i,  1555). 

Thymol:  injurious  to  oxydases  (Kastle  and  Loeven- 
hart) and  saliva-diastase  (Schlesinger,  Pugliese) 
and  markedly  so  to  zymase  (B  u  c  h  n  e  r)  and  chymosin 
(Freudenreich).  It  does  not  weaken  the  action  of  the  more 


ACTIVATORS,  PARALYbORS  AND  POISONS  119 

concentrated  trypsin  solutions   (Kaufmann,    H.,  1903,  39, 
434)  or  that  of  yeast-maltase  (E  .  Fischer). 

Maltose    retards  peptic  digestion  (Sailer  and  F  a  r  r) . 

Salicylic    acid:      stated  to   have   a  weakening   effect 

on   pepsin   and   trypsin   and   also   on   lipase.     Autolytic   action 

and  that  of  xanthine-oxydase  (B  u  r  i  a  n  ,    H.,   1905,  43,  494) 

are,  however,  accelerated. 

Hydrocyanic  acid.  Whilst  this  acid  has  proved 
itself  an  extremely  powerful  poison  towards  catalase,  its  deleterious 
action  on  other  enzymes  is  considerably  weaker  and  sometimes 
very  faint.  Zymase  is,  indeed,  completely  inactivated,  but  this 
action  is  reversible  (B  u  c  h  n  e  r) .  Also,  according  to  F  u  1  d 
and  S  p  i  r  o  ,  chymosin  is  not  injured  and  the  same  is  the  case 
with  pepsin.  The  decomposition  of  polypeptides  by  erepsin  is 
accelerated  by  small,  but  inhibited  by  larger,  quantities  of 
potassium  cyanide  (Abderhalden,  Caemmerer  and 
Pinkussohn,  H.,  1909,  59,  293).  In  1%  solution,  hydro- 
cyanic acid  weakens  but  does  not  destroy  the  proteolytic  enzyme 
of  yeast-juice  (G  e  r  e  t  and  H  a  h  n  ,  Chem.  Ber.,  1898,  31,  202). 
The  sensitiveness  of  catalase  towards  hydrocyanic  acid  is 
shown  by  Senter's  results  which,  with  those  obtained  with 
certain  other  organic  poisons  are  given  below.1  The  concen- 
trations required  to  diminish  the  velocity  of  reaction  to  one-half 
are: 

Paralysor.  Grm.-mol.  per  litre. 

HCN  1/1000000 

Phenylhydrazine  1/20000 

Aniline  1/400 

A  1  k  a  1  oi  d  s.     The  older  literature  on  this  subject  is  given 
by  N  a  s  s  e    (Pflug.  Arch.,  1875,  11,  159)  and  in  the  work  of 

1Faitelowit2   has   arranged  various  paralysors,   according  to  the 
concentrations  in  which  they  affect  milk-catalase,  in  the  following  order: 

i  n 

HCN  H2C2O4 

KCN  HNO3 

KCNS  Ba(NO3)2 

HgCl2  HC1 

H2S  CHsCOOH 
Hg(CN)2 


120  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Chittenden,  to  whom  a  very  thorough  investigation  is  due. 
In  general,  alkaloids  retard  enzyme  action,  but  not  very 
strongly  (Chittenden;  Gockel);  cf.  also  L  a  q  u  e  u  r 
(Arch.  f.  exp.  Path.,  1906,  55,  240)  who  studies  especially  the 
effect  of  quinine  on  enzymes.  According  to  the  latter  author, 
the  autolytic  enzyme  and  blood-oxydase  are  the  most  strongly 
inhibited  by  quinine.  A  s  h  e  r  observed  a  deleterious  action 
of  quinine  preparations  on  peptic  digestion.  Oxydases  appear 
to  be  especially  sensitive  to  alkaloids  (R  o  s  e  n  f  e  1  d) .  Hor- 
denine  sulphate  retards  peptic  and  tryptic  digestion  (Camus), 
but  not  the  action  of  maltase,  invertase  or  lipase. 

Of  practical  importance  for  the  technique  of  enzymology 
are  the  investigations  dealing  with  the  action  of  alcohol 
on  enzymes : 

Small  quantities  of  alcohol  have  an  accelerating  influence 
on  lipase  (G  i  z  e  1 1 ,  Zentralbl.  f.  Physiol.,  1905,  19,  769,  851) 
but  in  other  cases  a  more  or  less  complete  inhibiting  action,  e.g., 
on  trypsin  (Gizelt,  loc.  cit.),  rennet  (R  e  i  c  h  e  1  and 
S  p  i  r  o  ,  Hofm.  Beitr.,  1905,  7,  485)  and  diastase.  Only  tyro- 
sinase  is  able  to  act  in  50%  methyl-ethyl  alcohol.  Inhibition 
by  alcohol  is  almost  always  reversible. 

Even  very  large  quantities  of  alcohol  are  withstood  for  a 
time  by  all  enzymes,  as  the  ordinary  precipitation  methods 
show.  If  the  alcohol  is  removed  after  precipitation  of  the  enzyme, 
the  latter  resumes  its  activity  (Schondorff  and  V  i  c  t  o  r  o  w  , 
Pflug.  Arch.,  1907,  116,  495). 

Like  the  activators,  inhibiting  agents  exert  their  action  by 
combining  partly  with  the  substrate,  partly  with  the  enzyme 
and  partly  with  the  activator.  Especially  often  where  the 
paralysors  are  acids  or  bases  must  these  actions  play  a  part. 
Also,  salts  of  the  heavy  metals  alter  the  state  of  solution  of 
colloidal  substrates  such  as  proteins,  starch,  etc. 

The  behaviour  exhibited  by  antiseptic  and  narcotic  media, 
such  as  toluene,  thymol  and  chloroform,  towards  living 
c  e  1 1  s  is  determined  principally  by  the  behaviour  and  the  altera- 
tions of  the  lipoidal  plasma-skin.  The  plasma  undergoes  change 
only  after  the  penetration  of  the  plasma-skin  by  the  narcotic. 

The  enzymic  actions  of  living  cells  are  influenced  in  various 
ways.  By  toluene  or  chloroform,  fermentation,  for  example, 
is  interrupted,  although  zymase  itself — as  is  shown  by  experi- 


ACTIVATORS,   PARALYSORS   AND  POISONS 


121 


ments  with  press  yeast-juice — is  not  injured  by  chloroform. 
On  the  other  hand,  yeast-cells  invert  cane-sugar  equally  quickly 
in  absence  or  presence  of  chloroform  or  toluene. 

H.  Euler  and  Beth  af  Ugglas  (H.,  1911,  70,  279) 
have  arrived  at  the  result,  that  the  activity  of  those  enzymes 
which  are  combined  with  the  plasma  in 
the  living  cell,  e.g.,  zymase,  is  annulled  by  narcotics, 
whilst  the  enzymes  occurring  free  in  the  cells,  as,  for  example, 
invertase,  are  not  influenced  by  these  substances. 

Euler  and  K  u  1 1  b  e  r  g  have  investigated  the  connection 
between  the  sensitiveness  to  poisons,  extractability,  and  relative 
quantities  of  the  enzymes  of  a  beer-yeast  called  "  H  "  from  a 
Stockholm  brewery  (H.,  1911,  73,  85),  the  results  being  given 
in  the  following  table : 


Zymase. 

Monilia- 
invertase. 

Maltase. 

Beer-yeast 
invertase. 

Living 

yeast 

Relative  velocity 
of  reaction  in8% 
sugar  solution 
Extractability  .  .  . 
Action  of  poisons: 
Chloroform  

Thymol 

1 

0 

inhibited 
completely 
ditto 

1  (to  2) 
0 

inhibited 
completely 
ditto 

1  (to  2) 
0 

inhibited 
completely 
considerable 

170 

slight 

scarcely  any 
weakening 
no  weaken- 

Toluene  

Weakening       on 
drying  

ditto 
20  :  1 

ditto 
25  :  1 

weakening 
considerable 
weakening 

ing 
ditto 

2  :  1 

Dried 

yeast 

Extraetability  .  .  . 

Action  of  poisons  : 
Chloroform.  .  . 

Toluene  

very  slight 

weakened  . 
weakened 

very  slight 
weakened 

very  in- 
complete 

weakened 

about  20% 

not  weak- 
ened 
not  weak- 

ened 

Concerning  the  chemical  compounds  presumably  formed  in 
the  poisoning  of  the  free  enzymes  we  have  no  data,  since  we 
do  not  even  know  the  nature  of  the  reacting  enzymic  substance. 
These  chemical  combinations  often  appear  to  be  only  loose 
ones;  the  ability  of  the  enzyme  to  regain  wholly  or  partially 
its  original  activity  after  removal  of  the  paralysor,  is  especially 
marked  with  "  poisoning  "  by  hydrocyanic  acid. 


122  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

If  the  paralysor  is  present  in  only  very  small  quantities, 
it  may  be  destroyed  by  any  oxidising  agent  present  or  by  other 
enzymes  without  external  action.  To  this  must  be  attributed 
the  spontaneous  re-activation  which  is  known  as  "  recovery." 

Analogies  to  known  chemical  processes  are  here  also  not 
lacking.  A  comparison  has  often  been  drawn  between  the  actions 
exerted  by  paralysors  on  catalase  and  on  B  r  e  d  i  g  ;s  so-called 
"  colloidal  metals  " — which  are,  indeed  oxides — and  other  oxidis- 
ing catalysts.  But  even  S  e  n  t  e  r  '  s  thorough  experiments, 
referred  to  above,  are  not  able  to  explain  to  what  chemical  changes 
the  active  oxygen,  the  action  of  which  is  here  in  question,  is 
subjected  by  the  paralysors.  Besides,  the  "  poisonings '"  with 
catalase  and  colloidal  platinum  follow  by  no  means  parallel 
courses.  The  principal  result  of  the  interesting  contributions  of 
H  6  b  e  r  (Pfliig.  Arch.,  1900,  82,  631)  and  of  Loevenhart 
and  K  a  s  1 1  e  (Amer.  Chem.  Journ.,  1903,  29,  397)  to'  this  sub- 
ject may  be  summed  up  in  the  sentence:  "  That  the  effect  of  any 
particular  substance  on  the  catalyser  can  be  explained,  in  the 
majority  of  cases  at  least,  upon  purely  chemical  grounds." 

In  many  respects  the  anti-bodies  correspond  with  the  inhibit- 
ing substances  here  considered  and  the  limits  of  the  term  anti- 
bodies are  determined  by  characteristics  somewhat  similar  to 
those  which  define  the  limits  of  the  enzymes  in  the  wider  field 
of  the  catalysts.  A  number  of  substances  which  retard  enzymic 
reactions  in  the  normal  organism  should  also  be 
termed  inhibiting  substances  in  contradistinction  to  the  anti- 
enzymes,  which  the  organism  forms  as  protective  substances 
after  the  introduction  of  foreign  enzymes. 

A  whole  group  of  inhibition  phenomena  are  to  be  attributed 
to  the  adsorption  of  enzymes.  To  such  phenomena  belongs 
especially  the  inhibition  of  trypsin,  rennet  and  invertase  by 
charcoal,  studied  by  S.  G.  H  e  d  i  n  and  by  A.  Eriksson. 
H  e  d  i  n  showed  that  the  retardations  caused  by  white  of  egg  and 
serum-albumin  are  analogous  to  those  produced  by  charcoal, 
that  is,  they  are  due  to  adsorption  phenomena.  To  this  group 
belong  therefore  all  the  non-specific  inhibiting  effects  of  serum 
which  were  formerly  ascribed  to  anti-bodies.  H  e  d  i  n  has 
collected  the  literature  on  this  subject  in  the  ninth  yearly 
volume  of  the  Ergebnisse  der  Physiologic  (1910)  (cf.  H.,  1911, 
72,  313). 


ACTIVATORS,  PARALYSORS  AND  POISONS  123 

The  data  are,  however,  too  incomplete  to  permit  of  a  critical 
classification  of  these  inhibiting  substances,  so  that  a  resume 
of  all  substances  referred  to  in  the  literature  as  anti-enzymes 
will  be  given  in  a  later  chapter. 

Reference  must  finally  be  made  to  those  inhibiting  effects 
observed  when  heated  enzyme  solutions  are  added  to  the  active 
enzymes. 

Such  retardation  has  been  observed: 

With  tryptase  by  Pollak  (Hofm.  Beitr.,  1904,  6,  95) 
in  the  digestion  of  gelatine. 

With  peptase  by  Schwarz  (Hofm.  Beitr.,  1905,  6,  524), 
according  to  whom  the  inhibitory  substance  resists  the  action  of 
heat  and  exists  ready-formed  in  fresh  solutions  of  the  enzyme. 

With  peptase,  rennet  and  taka-diastase  by  Cramer  and 
Beam  (Proceedings  of  the  Physiol.  Soc.,  June  2,  1906,  see 
Journ.  of  Physiol.,  1906,  34,  xxxvi;  Biochem.  J.,  1907,  2,  174), 
who  heated  the  enzyme  solutions  to  50-60°;  at  100°  the  inhibit- 
ing substance  is  destroyed. 

With  invertase  by  A.  Eriksson  (H.,  1911,  72,  330),  who 
assumes  the  existence  of  an  inhibiting  agent,  resistant  to  heat, 
in  invertase  solutions. 

Here  belong  also  the  results  obtained  by  P  o  r  t  e  r  (Biochem. 
Z.,  1910,  25,  301)  with  peptase,  tryptase,  rennet,  lipase,  saliva- 
amylase,  amygdalase  and  taka-diastase.  In  contact  with  collo- 
dion membranes  these  enzymes,  with  the  exception  of  taka- 
diastase,  lose  their  activity,  all  of  them  except  saliva-amylase 
then  exhibiting  a  retarding  action  on  the  corresponding  enzymes. 


CHAPTER  IV 
CHEMICAL   DYNAMICS    OF    ENZYME    REACTIONS 

THE  relations  required  by  chemical  dynamics  for  the  simplest 
cases  of  catalytic  reactions  are  found  to  be  fulfilled  by  enzymic 
processes  to  very  varying  extents.  In  some  cases  the  time-law 
for  unimolecular  reactions  and  the  proportionality  between  veloc- 
ity of  reaction  and  amount  of  catalyst  are  closely  followed. 
But  in  the  majority  of  instances  the  experimental  data  are  in 
agreement  with  the  doctrine  of  reaction  only  within  a  limited 
region  of  concentration,  and  not  a  few  reactions  will  be  described 
for  which  no  simple  theoretical  representation  has  yet  been 
found  possible. 

The  arrangement  of  the  various  enzymic  processes  according 
to  the  mathematical  expressions  which  they  follow  is  apparently 
the  simplest  method  to  adopt.  But  this  can  only  be  carried 
out  at  the  expense  of  brevity  and  clearness  since,  as  already 
mentioned,  the  kinetics  of  the  action  of  one  and  the  same  enzyme 
may  be  altered  completely  by  change  of  the  external  conditions. 
Separate  treatment  will  therefore  be  given  for  each  of  the  more 
important  enzymes,  beginning  with  the  hydrolysing  enzymes 
and  passing  on  to  the  fermentation  enzymes,  oxydases  and 
catalases. 

The  opportunity  must  not  be  neglected  of  emphasising  the 
necessity  of  a  critical  examination  of  the  numerical  data  of  the 
quantitative  results  given  below.  Even  in  the  most  favourable 
cases,  where  a  chemically  individual  substrate  has  been  employed, 
the  solutions  dealt  with  not  only  contained  a  chemically  unknown 
catalyst  in  unknown  concentration,  but  were  also  contaminated 
with  the  foreign  constituents  of  the  enzyme  preparation  and,  in- 
deed, with  substances  which,  sometimes  even  in  minimal  amounts, 
might  exert  a  deciding  influence  on  the  course  of  the  reaction  to 
be  observed.  In  short,  one  is  in  the  doubtful  position  of  making 

124 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       125 

quantitative  observations  on  a  system  insufficiently  investigated 
qualitatively. 

It  might  then  be  asked:  Are  quantitative  results  obtained 
with  enzyme  solutions  of  any  value  at  all  and  is  any  detailed 
treatment  of  them  advisable?  If  the  observations  made  on  a 
preparation  are  not  very  comprehensive  and  if  the  experimental 
conditions  are  varied  only  inconsiderably,  the  value  of  a  physico- 
chemical  investigation  of  an  enzyme  is,  indeed,  small.  But 
our  knowledge  of  enzymes  has  been  widely  extended  by  a  number 
of  studies  of  the  kinetics  of  reaction,  which,  especially  when 
considered  as  a  whole,  have  elucidated  the  general  relations  of 
enzyme  action  and  have  furnished  valuable  aid  in  the  elabora- 
tion of  enzymological  methods. 

Before  the  experimental  results  are  examined  in  detail,  it 
will  be  well  to  consider  the  theoretical  principles  to  be  applied 
in  judging  these  results. 

THEORETICAL   PRINCIPLES    OF    ENZYMIC   DYNAMICS 

A  knowledge  of  the  law  of  mass  action  may  be  assumed. 
This  states  that  tlie  active  mass  of  a  substance  is  proportional 
to  its  osmotic  pressure  and  hence  (within  rather  narrower  limits) 
to  its  concentration.  If  a  single  type  of  molecule  A  with  a  con- 
centration CA  is  changed  by  a  chemical  reaction  into  new  sub- 
stances, without  the  concentration  of  any  other  kind  of  mole- 
cule present  being  appreciably  altered,  then  —  for  given  external 
conditions  of  temperature,  pressure  and  medium  —  the  quantity 
of  substance  dCA  changed  in  every  interval  of  time  dt  per  unit 
of  volume  will  be  given  by  the  equation: 


(la) 


where  kf  is  a  constant,  termed  the  velocity  constant 
(also  reaction  constant)  of  the  process. 

This  constant  kr  retains  —  and  on  this  emphasis  must  be  laid  — 
its  value,  undiminished,  no  matter  how  far  the  reaction  has  pro- 
ceeded or  what  initial  concentrations  may  be  chosen. 

On  the  other  hand,  the  velocity  v,  that  is,  the  amount 
of  substance  changed  per  unit  of  time  and  in  unit  volume,  is 


126  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

dependent  on  (proportional  to)  the  concentration  CA.  It  assumes 
the  same  numerical  value  as  k',  if  CA  =  1,  i.e.,  if  the  substance 
transformed  amounts  to  one  grm.-mol.  per  litre  and  is  in  some 
way  maintained  at  this  concentration. 

This  simplest  equation  allows,  therefore,  of  the  representa- 
tion of  a  chemical  process  which  proceeds  (practically)  completely 
in  one  direction. 

Integration  of  (la)  gives  the  constant  for  so-called  u  n  i  m  o- 
lecular  reactions: 


A/=.Zn—    or    fc  =    .log--       .    .    .     (16) 
t      a—x  t      &  a—  x' 

if  we  indicate  decimal  logarithms  by  log  and  hence  make 
/b  =  0-4343  A:'.1 

An  example  of  an  enzyme  reaction  in  which  molecules  of 
only  one  kind  undergo  change,  is  the  decomposition  of  hydrogen 

1  If  a  reaction  proceeds  so  that  equimolecular  quantities  of  t  w  o  sub- 
stances combine  to  form  the  product  of  the  reaction,  i.e.,  according  to  the 
scheme, 

A  +£-><?, 

then,  if  the  initial  concentrations  are  a  and  6,  and  x  denotes  the  amounts 
of  these  two  substances  (and  hence  the  concentration  of  C)  changed  after 
time  t,  the  velocity  at  any  moment  is  proportional  to  the  concentrations  of 
the  two  reacting  substances;  thus, 


or,  if  the  two  substances  have  originally  the  same  comcentration,  a, 

£-*<»-*)•. 

This,  the  simplest  case  of  a  so-called  bimolecular    reaction, 
corresponds  with  the  integral 

*  =  -—-. 

at  a—x' 

Enzymic  processes  proceeding  according  to  the  equation  for  bimolecular 
reactions,  are  as  yet  unknown. 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       127 

peroxide  by  catalase;  the  tables  given  on  p.  216  show  that  the 
values  of  k  are  constant  within  the  limits  of  experimental  error. 

The  above  equation  is  also  confirmed  in  numerous  cases 
where,  in  addition  to  a  dissolved  substrate,  water  takes  part 
in  the  reaction.  Since  the  water  is  usually  present  in  large 
excess  compared  with  the  dissolved  substance,  its  concentration 
may  be  regarded  as  constant;  so  that  here  also  only  molecules  of 
a  single  kind  undergo  change.  Indeed  the  first  example 
of  the  validity  of  the  unimolecular  reaction  law  was  that  of  the 
inversion  of  cane-sugar  by  acids  (W  i  1  h  e  1  m  y ,  1850).  Hud- 
son's recent  measurements,  the  results  of  which  are  given 
on  p.  160,  show  that  the  same  law  holds  for  the  hydrolysis  of 
this  sugar  by  invertase. 

It  may  here  be  pointed  out  that  the  law  of  mass  action,  on 
which  the  whole  of  chemical  kinetics  depends,  may  be  derived 
from  the  two  fundamental  laws  of  thermodynamics,  and  is 
hence  independent  of  our  present  molecular  kinetic  conceptions. 
That  the  law  of  mass  action  underlies  all  chemical  processes, 
cannot  therefore  be  doubted;  the  only  question  is  as  to  when  and 
how  far  the  assumptions,  according  to  which  it  can  be  expressed 
in  the  above  simple  form,  are  valid.  If  we  find  deviations  from 
the  simple  formulae  to  which  the  law  of  mass  action  leads,  we 
have  to  enquire  which  of  the  assumptions  are  not  fulfilled  under 
the  experimental  conditions  chosen. 

CATALYSIS 

The  hydrolysis  of  cane-sugar,  as  is  well  known,  proceeds 
with  extreme  slowness  in  pure  water;  the  velocity  is  considerably 
increased  only  when  the  solution  contains  an  acid  (or  an  enzyme, 
invertase),  in  addition  to  the  sugar.  So  far  as  we  can  determine 
by  titration,  the  concentration  of  the  added  acid  does  not  change 
during  the  hydrolysis.  Also  the  law  for  unimolecular  reactions 
holds  equally  well  for  a  solution  containing  either  0-001  or  0-1 
grm.-mol.  of  acid  per  litre,  the  sole  change  (so  long  as  we  remain 
within  the  region  of  dilute  aqueous  solutions)  being  in  the  nu- 
merical value  of  the  reaction  constant  k.  A  catalyst  is  there- 
fore, as  mentioned  at  the  outset,  to  be  defined  asasubstance 
which,  without  being  consumed  in  the  reac- 
tion, alters  the  velocity  with  which  a 


128  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

reaction     attains     its     position    of     equilib- 
rium.1 

In  1884  Arrhenius  made  the  important  discovery,  that 
the  catalytic  action  of  different  acids  runs  parallel  with  their 
conductivity  or,  more  accurately,  with  the  amount  dissociated. 

This  law,  which  has  been  often  confirmed  and  is  of  the  widest  signifi- 
cance, can  be  expressed  by  saying  that  different  acids  catalyse  hydrolytic 
reactions  in  proportion  to  the  concentration  of  the  hydrogen-ions  of  their 
solutions.  This  fact  is  often  stated  in  the  literature  in  such  a  way  as 
to  imply  that  the  hydrogen-ions  alone  are  the  catalysing  agent  and  that 
they  function  as  a  kind  of  contact-substance.  But  such  a  representation 
by  no  means  corresponds  with  the  chemical  facts.  Rather  must  it  be 
supposed  that,  by  the  catalysing  acid  the  concentration  of  the  ions  effect- 
ing the  reaction  is  increased  (E  u  1  e  r  ,  Zeitschr.  f.  physikal.  Chem., 
1901,  36,  681) 

The  supposition  that  combination  of  the  catalyst  with  the 
substrate  yields  the  molecules  which  carry  on  the  reaction,  has 
already  received  general  acceptance  in  enzymology.  Of  the 
authors  who,  on  the  basis  of  their  own  investigations,  have  expressed 
themselves  in  this  sense,  mention  need  only  be  made  of :  K  a  s  1 1  e 
and  Loevenhart,  Bach,  Hanriot,  A.  Brown,  H. 
Brown  and  Glendinning,  Bodenstein,  Henri, 
Medwedew,  Hedin,  Armstrong  and  B  a  y  1  i  s  s  . 

In  many  enzymic  reactions,  compounds  between  enzyme 
and  substrate  seem  to  occur  to  a  far  greater  extent  than  is  the 
case  in  catalytic  hydrolyses  by  acids;  yet  in  no  instance  has  it 
been  determined  what  proportions  of  the  total  quantities  of 
enzyme  and  substrate  present  combine  during  the  reaction. 

It  would  lead  too  far  to  indicate  the  reasons  which  have 
caused  the  various  investigators  to  assume  a  combination  of 
enzyme  with  substrate.  We  shall  only  indicate  briefly  the 
mathematical  formulation  of  the  hypothesis  in  question. 

In  his  researches  on  the  inversion  of  cane-sugar,  Henri  found 
that  the  reaction  constants  of  the  first  order  increased  considerably 

1  This  holds  for  all  those  cases  where  a  substance  does  not  catalyse  its 
own  transformation,  as  is,  for  example,  the  case  with  the  formation  of 
lactones  from  y-  and  8-hydroxy-acids,  the  hydro xy-acid  here  accelerating 
the  lactone-formation  proportionately  with  the  amount  of  its  dissociated 
portion  (auto- catalysis). 


CHEMICAL  DYNAMICS   OF  ENZYME  REACTIONS       129 

(cf.  p.  158),  and,  in  order  to  obtain  a  mathematical  expression  of  his 
results,  he  employed  (Zeitschr.  f.  physikal.  Chem.,  1902,  39,  194)  a 
method  given  by  0  s  t  w  a  1  d  (Lehrbuch  der  allgem.  Chemie,  II,  2,  265). 

If  a  reaction  is  accelerated  by  a  substance  having  the  concentration 

p,  the  expression  k(a  —x)  is  to  be  multiplied  by  (1  +ep),  e  being  a  constant. 

For  the  case  in  which  the  product  of  the  reaction  is  the  accelerating 

agent,  the  Velocity  will  hence  increase  in  the  ratio  1  :(l+e  —  j,  i.e., 

in  proportion  to  the  transformed  part  —  of  the  substrate.  The  equation 
therefore  becomes : 

f-for(l+.|)(a^) (Ic) 

On  integration,  this  gives: 

....     (Id) 


or 

Calculation  of  several  series  of  experiments  on  the  action  of  invertase 
gave,  for  the  new  constant  e,  values  approaching  1.  Thus,  if  e  =  l, 
the  equation  becomes 


or 


Shortly  afterwards  Bodenstein  subjected  Henri's  numbers 
to  a  re-examination,  making  the  assumption  that  both  the  cane-sugar  a 
and  its  hydrolytic  products  weaken  the  invertase,  the  former  more 
strongly  than  the  invert-sugar.  From  this  he  derived  the  formula: 


...     (3) 

(Cf.    Henri,    Lois  gene"rales  de  Faction  des  diastases,  Paris,   1903, 
p.  77  et  seq.) 

Here  i  indicates  the  quantity  of  invert-sugar  previously  added,  E 
denotes  the  quantity  of  enzyme  and  m  and  n  are  constants  expressing 
the  specific  weakening  of  the  invertase  by  cane-sugar  and  invert-sugar 
respectively.  Bodenstein  chose  for  these  constants  the  values 


130  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

m=2  and  n  =  l,  thus  indicating  the  stronger  action  of  the  cane-sugar. 
If  the  solution  originally  contains  no  reaction-product  (i  =0),  his  formula 
simplifies  to  the  following: 


While  this  equation  corresponds  satisfactorily  with  observations  on 
moderately  dilute  solutions,  the  numbers  obtained  by  Henri  with 
dilute  solutions  are  not  in  agreement  with  it. 

Henri  has  therefore  deduced  another  expression  on  the  assumption 
that  both  the  cane-sugar  and  the  invert-sugar  (especially  the  fructose) 
combine  with  the  enzyme  (Lois  generates,  p.  85  et  seq.,  and  C.  R.,  1902, 
135,  916). 

Of  the  original  quantity  of  substance  a  let  x  molecules  be  hydrolysed, 
so  that  a—  x  molecules  remain.  Further,  let  the  quantity  of  enzyme 
be  E  and  X  the  quantity  of  it  which  is  free  and  Z  and  Y  the  amounts 
which,  at  the  time  t,  are  combined  with  the  cane-sugar  and  invert-sugar 
respectively. 

Between  enzyme  and  substrate  on  the  one  hand,  and  enzyme  and 
products  of  reaction  on  the  other,  equilibria  must  set  in  in  accordance 
with  the  law  of  mass  action.  Hence  for  these  two  equilibria  the  follow- 
ing relations  hold  (in  the  case  where  1  mol.  of  enzyme  unites  with  1  mol. 
of  substrate)  • 

X(a-x}=—  .Z,  (5) 

m      ' 

and 

**=i-r,     ........   (6) 

where  m  and  n  are  equilibrium  constants. 
Further,  for  the  total  quantity  of  enzyme 

E=X  +  Y+Z  .........     (7) 

From  these  Henri  calculated  the  quantity  of  free  enzyme  X 
and  that  of  the  compound  sugar-enzyme  Z: 

E 
X= 


l+m(a-x)+nx' 
and 


Z  __ 

l+m(a-x)+nx' 


Only  two  assumptions  can  now  be  made: 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       131 

1.  The    free    portion    of   the    enzyme    acts    on    the 
sugar;    in  this  case  the  velocity  is  proportional  to  the  quantity  of 
free  enzyme  and  to  the  quantity  of  cane-sugar,  i.e.,  to  X  and  to  a—x. 
Hence 

^  =  const.  X(a-x) (10) 

If  X  is  substituted  in  accordance  with  Eq.  (8),  this  gives 

dx      const.  E(a  —x) 
dt~l+m(a-x)+nx 

2.  If,  on  the  other  hand,  the  reaction  is    effected    by    the 
complex,    enzyme-sugar,    the  velocity  will  be  proportional 
to  the  concentration  of  these  molecules,  i.e.,  to  Z.     From 

7=  const.  Z, 


is  obtained,  from  Eq.  (9), 

dx    const.  m.E(a  —x) 
dt  ~  1  +m(a  —x)  +nx  ' 


(12) 


So  that  both  assumptions  lead  to  one  and  the  same  expression  for 
the  velocity  of  reaction. 

On  the  supposition  that  complexes  are  formed  consisting  of  1  mol. 
enzyme,  p  mols.  of  substrate  and  q  mols.  of  the  various  products  of  the 
reaction,  Arrhenius  (Immunochemistry,  p.  60)  gives  a  still  more 
general  form  for  the  above  expression. 

For  every  single  such  molecule  a  formula  is  obtained  of  the  form: 


.....     (13) 
where 


If  we  assume  that  sZ  is  small  compared  with  a  and  x,  we  obtain 

Z=KfX(a 
so  that 


If  the  first  term  of  the  denominator,  1,  is  small  with  respect  to  the 
terms  under  the  summation  sign,  Bodenstein's  formula  is  obtained 
if  we  make  a  =  1  and  assume  that  there  are  two  terms  under  the  s,  one 
in  which  p  =  I  and  q  =0  and  a  second  in  which  p  =0  and  q  =  l. 


132  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

In  a  preliminary  communication  published  two  years  later,  Henri 
(Zeitschr.  f.  physikal.  Chem.,  1905,  51,  19)  outlines  a  new  theory  of 
enzyme-action  which  takes  account  of  the  colloidal  condition  of  enzymes. 
According  to  the  law  of  distribution,  the  dissolved  body,  sugar  for 
example,  must  be  distributed  between  the  aqueous  solution  and  the 
colloid,  the  velocity  of  reaction  being  determined  by  the  concentration 
of  the  sugar  in  the  colloid.  In  this  way  Henri  explains  the  similarity 
between  the  ordinary  adsorption  curves  and  those  representing  the 
influence  of  the  concentration  of  the  cane-sugar  on  the  velocity  of  inver- 
sion by  invertase. 

None  of  these  formulae  and  theories  have  been  confirmed  in  a  manner 
free  from  objection. 

As  has  been  mentioned  previously,  the  hydrolysis  of*  cane- 
sugar,  esters  and  other  similar  substances  is  accelerated  by  acids, 
the  acceleration  being  proportional  to  the  concentration  of  the 
hydrogen-ions  in  the  solution.  If,  as  is  usually  done,  strong  acids 
are  used  in  very  low  concentrations,  there  is  approximate  pro- 
portionality between  the  concentration  of 
the  catalyst  and  the  velocity  of  reaction, 
since  the  electrolytic  dissociation  of  the  acid  is  virtually  complete. 

Such  proportionality  between  concentration  of  the  catalyst  and 
the  velocity  of  reaction  is  found  to  hold  in  numerous  enzyme 
reactions  within  quite  wide  limits  of  concentration.  This  is  the 
case,  for  example,  with  the  actions  of  the  lipases  (p.  146  et  seq.), 
catalases  (p.  216),  invertase  (p.  160)  and  erepsin  (p.  189). 

An  exception  to  this  very  simple  relation  has,  however,  been 
known  for  a  long  time.  The  amounts  of  protein 
digested  by  pepsin  in  a  definite  time  are 
proportional,  not  directly  to  the  quantities 
of  pepsin,  but  to  the  square  roots  of  these. 
This  is  the  law  which  was  enunciated  byEmil  Schiitz  in 
1885  (H.,  1885,  9,  577). 

The  validity  of  this  rule  has,  indeed,  often — even  in  recent 
times — been  contested.  But  the  reliability  of  the  numerous 
experiments,  made  by  different  methods  (cf.  p.  176  et  seq.) 
and  confirming  S  c  h  ii  t  z  's  numbers — we  are  referring  now 
exclusively  to  pepsin  action — cannot  be  doubted.  Hence  for 
peptic  digestion,  at  any  rate  in  the  first  third  of  the  reaction, 
S  c  h  ii  t  z  's  rule  is  obeyed,  and  we  are  met  with  the  problem 
of  explaining  this  experimental  relation  on  the  basis  of  the  doc- 
trine of  chemical  dynamics. 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS      133 

Arrhenius  (Medd.  Nobel  Inst.,  1908,  1,  No.  9)  has 
deduced  S  c  h  ii  t  z  's  rule  theoretically  in  the  following  way  : 

In  order  to  ascertain  the  circumstances  which  condition 
S  c  h  ii  t  z  's  rule,  we  proceed  as  follows  :  If  the  amount  of 
substance  transformed  is  indicated  by  x  and  the  time  by  t,  the 
rule  states  that  : 

x  =  KiVT    or      x2  =  ^2t  ......     (15) 

On  differentiation  this  gives: 


or: 

dx_*l\_  ,     } 

dt~  2    x' 

So  that,  for  S  c  h  ii  t  z  '  s  rule  to  hold,  it  is  a  necessary 
and,  as  may  easily  be  seen,  sufficient  condition  that  the  velocity 
of  reaction  shall  be  inversely  proportional  to  the  quantity  of 
substance  transformed,  i.e.,  to  x.  Since  the  rule  is  only  obeyed 
during  the  initial  stages  of  the  reaction,  this  proportionality  must 
only  be  assumed  for  the  first  part  of  the  change. 

Such  proportionality  may  be  brought  about  as  follows:  The 
velocity  of  reaction  —  in  the  case  when  only  one  molecule  of  each 
of  the  reacting  bodies  goes  into  the  product  of  the  reaction  —  is 
proportional  to  the  product  of  the  concentrations  of  the  reacting 
substances.  At  the  beginning  of  the  reaction,  such  small 
quantities  of  the  bodies  are  transformed  that  generally  their 
total  quantities  may,  with  sufficient  accuracy,  be  regarded  as 
constant.  It  is  this  that  limits  the  validity  of  Schiitz's  rule 
to  the  beginning  of  the  reaction.  Now  the  active  quantity 
(M)  of  one  of  the  reacting  bodies  must  be  inversely  proportional 
to  the  amount  changed,  i.e.,  to  the  quantity  of  new  product 
(x),  so  that: 

,,     const.  ,, 

M  =  --     or    MX  =  const. 

x 

This  is  evidently  the  case  if  a  chemical  equilibrium  is  set  up 
between  the  new  product  and  one  of  the  reacting  bodies,  on  the 
one  hand,  and  a  compound  of  them  in  almost  constant  quantity, 
on  the  other.  Such  a  case  is  already  known  in  the  saponifying 
action  of  ammonia  on  an  ester,  e.g.,  ethyl  acetate.  The  one 


134  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

reacting  body  here  is  the  ion  OH  and  the  quantity  of  this  ion 
(Mon)  is  diminished  by  the  NH4-ions  (amount,  zNH4)  of  the 
ammonium  acetate  formed  during  the  reaction  according  to  the 
equation  : 

4  =  Cl  (NNH4OH  +  NNH3)  , 


where  NNH4oH  and  NNH3  denote  the  amounts  of  the  OTUOH- 
and  NHs-molecules  respectively.  The  volume  remains  constant 
during  the  reaction  and  the  last-named  quantity  may  be  regarded 
as  constant,  so  long  as  the  beginning  of  the  reaction  is  alone 
considered. 

A  closer  investigation  of  this  case  by  Arrhenius  has 
revealed  distinctly  an  analogy  to  the  course  of  peptic  digestion. 

Arrhenius  considered  a  system  in  which  ammonia  in  an 
initial  quantity  A  acts  on  ethyl  acetate,  and  he  assumed,  for 
the  sake  of  simplicity,  that  the  amount  of  ethyl  acetate  is  so 
large  that  it  is  not  changed  appreciably  by  the  reaction  but  may 
be  regarded  as  constant  (P);  that  is,  the  ethyl  acetate  must 
be  present  in  considerable  excess.  If  now  x  mols.  of  ammonium 
acetate  —  which  with  the  high  dilution  that  we  assume  may  be 
considered  as  completely  decomposed  into  NEU-  and  CHsCC^- 
ions  —  are  formed  at  the  time  t,  (a—x)  mols.  of  ammonia  will  be 
present  at  the  same  time.  Owing  to  the  slight  dissociation  of 
ammonia,  only  a  very  small  fraction  of  it  is  changed  into  NH4- 
and  OH  -ions,  so  that  it  is  sufficiently  exact  to  denote  the  amount 
of  non-dissociated  ammonia  by  (a—x).  Even  in  presence  of  a  very 
small  quantity  of  ammonium  salt,  the  NH4-ions  from  the  ammonia 
may  be  neglected  in  comparison  with  those  formed  by  the  salt, 
and  the  amount  of  NH4-ions  may  be  taken  as  x.  On  this  assump- 
tion the  first  moments  of  the  reaction  must  be  disregarded,  as 
then  no  ammonium  salt  or  but  very  little  is  present.  The  con- 
centration of  the  hydroxyl-ions,  q,  is  then  determined  by  the 
following  equation: 

q.x  =  K2(a-x), 

where  K%  denotes  the  dissociation  constant  of  the  ammonia. 

The  velocity  of  reaction  on  hydrolysis  is  now  proportional 
to  the  quantity  of  hydroxyl-ions  (q)  and  to  the  amount  of  ethyl 
acetate  (P),  that  is: 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       135 


where  #2  and  x  are  constants  and  xP  may  be  regarded  as  a  new 
constant,  since  the  value  of  P  does  not  change  in  any  single 
experiment 

On  integration,  the  last  equation  gives: 


F(x)=aln- x  = 

a  —  x 


(18) 


The  following  table,  taken  from  that  given  byArrhenius, 
contains  the  result  of  a  duplicated  experiment  in  which  a  given 
quantity  of  ammonia  acted  on  0-66-normal  ethyl  acetate  at 
14-8°. 

Mean  value  of  fcP  =  21. 


Percentage  of  ammonia  converted. 

1 

•cP. 

Observed.  1 

Calculated. 

1 

17-5 

19-4 

17-4 

2 

25-5 

25-2 

22-0 

3 

30-7 

30-6 

21-2 

5 

38-5 

38-5 

20-9 

10 

51-2 

51-3 

20-9 

15 

59-6 

59-7 

20-9 

22 

67-5 

68-0 

20-6 

30 

74-5 

74-7 

21-2 

50 

84-8 

85-0 

20-8 

70 

91-1 

90-7 

21-6 

100 

95-3 

95-3 

21.  1 

1  Mean  of  two  experiments. 

As  will  be  seen,  formula  (18)  is  in  excellent  agreement  with 
the  observed  results. 

The  analogy  between  the  hydrolysis  of  esters  by  ammonia 
and  the  digestion  of  protein  by  pepsin  is,  according  to  A  r  r  - 
h  e  n  i  u  s  ,  as  follows : 

In  the  decomposition  of  protein,  albumoses  and  peptones  are 
formed.  Most  of  the  pepsin  is  fixed  by  the  products  of  the 
reaction,  so  that  the  following  equilibrium  is  set  up: 

[Free  pepsin]  X  [products  of  reaction]  =  const,  [combined  pepsin]. 

The  quantity  of  free  pepsin  is,  approximately,  inversely 
proportional  to  the  amount  of  the  reaction  products,  x.  This 
holds  as  soon  as  so  much  reaction-product  is  formed  that  the 


136  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

greater  part  of  it  is  not  combined  with  pepsin,  whilst  the  greater 
part  of  the  latter  is  combined. 

Further,  the  amount  of  free  pepsin  is  evidently  proportional 
to  the  quantity  of  pepsin  employed.  Hence,  if  we  indicate  the 
concentration  of  the  enzyme  (pepsin)  by  [E],  the  unaltered  pro- 
tein by  (a—x)  and  the  products  of  digestion  by  x,  we  obtain: 


(19) 


and  consequently,  if  we  express  the  amount  of  hydrolytic  products 
as  fractions  of  1000: 

1000(7nlOOO-Zn  unaltered  protein)—  digested  protein  =  K[E]t.  (20) 

In  reality,  peptic  digestion  corresponds  remarkably  well 
with  Arrhenius's  formula  and  it  is  because  the  latter  holds 
over  wider  concentration-limits  of  enzyme  and  substrate  that  the 
work  of  Arrhenius  on  Schiitz's  rule  has  been  here  re- 
ferred to  at  length. 

Whether,  indeed,  the  above  considerations  take  account  of 
all  the  facts  and  factors  essential  to  peptic  digestion,  e.g.,  the 
combination  of  hydrochloric  acid  (cf  .  Jastrowitz,  Biochem. 
Z.,  1907,  2,  157),  cannot  at  present  be  easily  decided.  In  par- 
ticular it  is  still  uncertain  what  proportion  of  the  pepsin  com- 
bines, under  definite  conditions,  with  the  substrate  or  reaction- 
products  and  what  proportion  with  the  hydrochloric  acid.  All 
that  can  be  said  is  that  the  spatial  configuration  of  the  participat- 
ing substances  plays  a  very  real  part.  This  can  be  seen  from 
the  influence  which  additions  of  optically  active  bodies  exert  on 
digestive  processes. 

It  would  be  of  interest  to  ascertain  the  concentrations  of 
hydrochloric  acid  for  which  pepsin-digestion  is  proportional  to 
the  concentration  of  the  enzyme. 

The  opportunity  must  be  taken  here  of  pointing  out  that, 
with  many  enzymic  reactions,  the  chemical  change  is  composed 
of  several  separate  processes  which  have  not  yet  been  studied 
singly;  this  is  the  case,  for  example,  with  the  hydrolysis  of  pro- 
teins or  starch  by  proteinases  or  amylases.  In  such  complex 
reactions  as  these  it  cannot,  of  course,  be  expected  that  the  laws 
of  chemical  dynamics  will  be  apparent  in  their  simplest  form. 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       137 

In  addition  to  the  above  processes,  the  fermentation  of  bioses 
by  yeasts  may  be  mentioned;  it  is  here  assumed  that  the  first 
phase  consists  of  a  hydrolysis  of  the  bioses  (cf .  E  u  1  e  r  and 
Lundeqvist,  H.,  1911,  72,  103). 

Finally,  as  has  been  done  by  C  .  E  n  g  1  e  r  and  R  .  O  .  H  e  r  - 
z  o  g  (H.,  1909,  59,  327),  enzymic  oxidations  may  be  represented 
as  coupled  or  induction  reactions.  If  M,  N  and 
P  are  three  substances  capable  of  reacting  as  follows: 

M+N  =  (MN)  react. 
P-\-N  do  not  react. 

M+ N+P  =  (MN)  +  (BN)  react. 

(where  the  bracketed  letters  indicate  the  substances  reacting 
with  one  another),  the  reaction  is  a  coupled  or  induced  one. 
Schilow  (Zeitschr.  f.  physikal.  Chem.,  1903,  42,  641;  cf. 
also  Luther  and  Schilow,  Zeitschr.  f.  physikal.  Chem., 
1903,  46,  777)  terms  the  substance  N,  taking  part  in  both  reactions, 
the  actor,  M  the  inductor  and  P  the  acceptor. 
According  to  E  n  g  1  e  r  and  H  e  r  z  o  g  ,  the 

oxydase  functions  as  inductor, 
oxygen  actor, 

oxidised  substance  acceptor. 

This  conception  opens  up  a  number  of  interesting  points. 

REVERSIBLE  REACTIONS 

Influence  of  the  Products  of  Reaction. 
Up  to  the  present,  hydrolytic  enzyme  reactions  have  been  treated 
as  though  they  proceeded  completely  in  one  direction  or,  more 
accurately,  as  though  the  opposite  synthetical  reaction  were  so 
inconsiderable  as  to  be  negligible.  We  know  that  the  hydrolysis 
of  cane-sugar  by  acids — the  classic  reaction  of  chemical  dynamics 
— is  practically  complete  under  ordinary  conditions  and  we 
should  at  first  expect  the  same  to  occur  also  with  enzymic 
hydrolyses.  But  recent  researches  of  Y.  Osaka  (Journ. 


138  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Coll.  of  Science,  Tokyo,  1908,  25,  1)  have  shown  that,  even 
with  cane-sugar,  if  sufficiently  concentrated  solutions  are  employed, 
the  equilibrium  is  apparent  just  as  it  usually  is  with  the  esters 
of  organic  acids.  The  change  which  such  a  reversible  system 
undergoes  with  lapse  of  time  is  the  difference  between  two  opposite 
actions. 

For  instance,  the  velocity  of  hydrolysis  of  the 
ester,  v  i  is  given  by  the  equation  : 

vi  =  ki  [ester], 

if,  as  usual,  we  indicate  the  concentration  by  [  ].  The 
velocity  of  formation  of  the  ester  V2  is  expressed  by: 

V2  =  ^[acid]  [alcohol], 

and  the  actual  resultant  velocity  in  a  system  not  in  equilibrium 
and  containing  ester,  acid,  alcohol  and  water,  is: 

v  =  vi  —  1>2  =  fci  [ester]  —  /^[acid]  [alcohol] 

or,  if  we  start  from  a  pure  ester  solution  of  the  concentration 
a  and  indicate  the  amount  changed  in  time  t  by  x: 

—  =  v  =  vi  —  V2  =  ki(a  —  x)—k2X2  .....     (21) 

The  ratio  between  the  two  velocity  constants  is,  as  was  shown 
byvan't  Hoff  (compare  Chapter  VI),  the  constant  of 
chemical  equilibrium,  so  that 

ki__       [ester] 


&2  [acid]  [alcohol]' 

When  the  constant  of  the  "  reverse  reaction  "  is  not  vanish- 
ingly  small,  the  time  course  of  the  total  reaction  v,  as  can  be 
easily  seen,  is  changed  if  the  product  of  the  reaction  x  is  previously 
added  to  the  system,  and,  in  general,  the  progress 
of  a  reversible  reaction  must  be  retarded 
by  addition  of  the  products  of  the  reaction. 

When  ki  and  k%  have  been  experimentally  determined,  it  is 
easy  to  calculate  how  v  changes  with  increasing  additions  of  x. 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS        139 

For  dilute  solutions  and  inorganic  catalysts  these  relations  have 
been  completely  worked  out.1 

With  enzyme  reactions  another  circumstance,  which  has  to 
be  considered,  complicates  matters  to  some  extent.  We  have 
already  discussed  the  assumption,  now  generally  accepted,  that 
the  enzymes  form  compounds  with  the  substrate  and  with  the 
products  of  reaction.  In  no  case  do  we  know  in  what  meas- 
ure an  enzyme  enters  into  such  combination,  but  we  assume 
that  these  compounds  exert  considerable  influence  on  the  time 
course,  so  that  the  velocity  of  an  enzymichydrolysis  is  altered 
by  addition  of  the  products  of  the  reaction  to  the  system,  not 
only  according  to  Eq.  (21),  but  also  by  combination  (inactiva- 
tion)  of  the  catalyst. 

This  influence  has  not  only  been  observed  qualitatively, 
but  has,  in  many  cases,  been  measured. 

Of  the  experiments  showing  the  retarding  influence  of  the 
hydrolytic  products,  that  quoted  by  W  .  K  ii  h  n  e  (Lehrbuch  der 
physiol.  Chem.,  1866,  p.  39)  is  one  of  the  earliest:  If  a  digestion 
solution,  filtered  from  excess  of  undigested  fibrin,  is  placed  in  a 
dialyser,  most  of  the  peptones  diffuse  into  water,  whilst  the 
pepsin  remains  behind.  When  the  pepsin  solution  is  restored 
to  its  original  volume  by  evaporation  and  to  its  initial  acidity, 
it  dissolves  almost  exactly  as  much  fibrin  as  was  previously 
dissolved.  It  is  consequently  the  peptones  which  hinder  the 
digestion. 

We  may  also  recall  the  investigation,  already  mentioned, 
of  Tammann  (H.,  1891,  16,  271)  which  showed  in  a  con- 
vincing manner  that  the  products  of  hydrolysis  generally  influence 
the  completeness  of  enzymic  reactions;  but,  as  he  says,  "  not 
only  by  removal  and  destruction  of  the  decomposition  products 
can  an  enzyme  reaction  be  rendered  complete,  as  the  same  end 
is  attained  by  repeated  additions  of  enzyme." 

Of  Tammann's  numerous  experiments,  the  following 
may  be  mentioned : 

To  different  solutions,  each  containing  0-51  grm.  of  amygdalin, 
were  added  equal  amounts  of  emulsin  and  varying  quantities 
of  saturated  benzaldehyde  solution,  all  the  solutions  being  then 

1  In  a  modification  of  this  equation  devised  for  reversible  enzyme  reactions, 
B.  Moore  (1906)  makes  an  attempt  to  take  account  of  the  activity  of  the 
enzyme. 


140  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

made  up  to  25  c.c.     When  the  final  state  was  reached  (at  20 °), 
the  following  quantities  of  amygdalin  were  decomposed: 

Volume  of  aldehyde  solution.  Percentage  of  amygdalin  decomposed. 

0  c.c.  20-3 

1  c.c.  18-8 
5  c.c.  14-7 

10  c.c.  11-31  Precipitation 

Solution  saturated  with  benzaldehyde.  5-7J      of  emulsin. 

Hydrocyanic  acid  has  a  still  more  marked  action : 
25  c.c.  of  solution  at  30°  contained  0-5  grm.  emulsin  and  0-001 
grm.-mol.  of  amygdalin: 

Hydrocyanic  acid  added.  Amygdalin  decomposed. 

0-0000  grm.-mol.  23-7 

0-0001    "  18-7 

0-0002    "  16-4 

,0-0003    "  12-1 

The  effect  of  the  third  product  of  the  decomposition,  glucose, 
is  much  less  marked,  as  is  shown  by  the  following  table,  due  to 
Auld  : 

Minutes.  Grm.  of  Glucose  added.  Amygdalin  hydrolysed. 

30  0-0  13-5% 

30  0-2  13-3 

30  0-75  11-8 

30  1-0  11-6 

In  the  further  investigation  of  this  influence  it  should  be 
remembered  that  emulsin  consists  of  several  specifically-acting 
enzymes. 

That  the  diastatic  hydrolysis  of  starch  gives  an  end  point 
which  is  influenced  by  the  sugar  formed,  is  pointed  out  by 
M  o  r  i  t  z  and  Glendinning  (Journ.  Chem.  Soc.,  1892, 
61,  689). 

Henri  has  made  numerous  experiments  on  the  effect  of 
added  glucose  and  fructose  on  the  inversion  of  cane-sugar,  the 
establishment  of  an  effect  of  this  kind  being  of  importance  for 
the  development  of  his  formula.  As  has  been  often  pointed  out, 
it  is  greatly  to  be  regretted  that  his  experimental  numbers  are 
considerably  distorted  owing  to  the  mutarotation  of  glucose, 
so  that  quantitatively  they  are  almost  valueless.  It  does  appear, 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       141 


however,  that  invert-sugar  •  lessens  the  velocity  of  inversion. 
The  following  table  is  compiled  from  Henri's  numbers 
(1  o  c  .  c  i  t  . ,  p.  202) : 

2  c.c.  of  enzyme  solution +50  c.c.  0-2-normal  cane-sugar  solution. 


t 

Without 
addition. 

X 

+0-3-normal 
invert-sugar. 

X 

a 

a 

99 

0-138 

0-072 

215 

0-301 

0-160 

299 

0-407 

0-224 

459 

0-594 

0-340 

586 

0-700 

0-417 

1202 

0-927 

0-672 

The  0  •  2-normal  cane-sugar  solution  which  is  also  0  •  3-normal 
with  respect  to  invert-sugar  was  therefore  hydrolysed  only  half 
as  rapidly  as  that  without  invert-sugar. 

This  retarding  influence  of  invert-sugar  seems  to  be  due 
exclusively  to  the  fructose,  as  is  shown  by  the  following  values  of 

cc 

—  given  by  H  e  n  r  i  . 


Time. 

0-2  N-cane-sugar. 

0  •  2  N-cane-sugar. 
+  0-2  N-glucose. 

0-2  N-cane-sugar 
+  0-2  N-fructose. 

0-2  N-cane-sugar. 
+  0-2  N-invert- 
sugar. 

75 

0-142 

0-144  , 

0-123 

0-119 

184 

0-385 

0-362 

0-317 

0-306 

275 

0-564 

0-532 

0-457 

0-446 

445 

0-798 

0-746 

0-672 

0-648 

605 

0-906 

0-878 

0-799 

0-794 

As  will  be  seen,  the  retardation  of  a  reaction  by  the  hydro- 
lytic  products  is  decidedly  specific.  That  this  is  the  case  was 
also  brought  out  very  clearly  in  a  table  given  by  E.  F.  A  r  m  - 
strong  (Proc.  Roy.  Soc.,  1904,  73,  516)  to  show  the  effect  of 
the  hexoses  in  delaying  the  hydrolysis  of  sugars.  Still  more 
striking  examples  of  the  specificity  of  such  retardations  will 
doubtless  be  obtained  from  the  investigation  of  the  polypeptides 
which  has  recently  been  taken  in  hand. 


142 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


K  ii  h  n  e  's  experiment  on  the  influence  of  the  decomposi- 
tion products  on  the  peptic  digestion  of  fibrin  has  already  been 
quoted.  B  a  y  1  i  s  s  has  made  experiments  on  the  retardation 
of  the  tryptic  digestion  of  casein  by  albumoses,  peptones  and 
amino-acids  (Arch.  Sci.  Biol.  St.  Petersburg,  1904,  11,  Supple- 
ment, pp.  261  et  seq.),  his  results  showing  that  the  amino-acids 
— glycine  and  leucine  were  examined — are  the  most  active  in 
this  respect. 

In  investigating  the  hydrolysis  of  dipeptides  (glycylglycine) 
by  erepsin  E  u  1  e  r  also  made  experiments  on  the  influence  of 
the  amino-acids  (glycine)  (H.,  1907,  61,  213).  In  this  case  it 
was  found  that  addition  of  glycine  has  only  a  subordinate  effect. 


0-  10  N-glycylglycine;  0-04  N-NaOH. 

0-05  N-glycylglycine;  0-10  N-glycine; 
0-04N-NaOH. 

Minutes. 

1000(a-a;). 

1000&.    • 

Minutes. 

1000(o  -x). 

lOOOfc. 

0 

955 

— 

0 

480 



8 

852 

6-25 

10 

414 

6-40 

16 

766 

6-00 

18 

372 

6-20 

25 

678 

5-95 

27 

329 

6-08 

0-20  N-glycylglycine;  0-05N-NaOH. 

0-10  N-glycylglycine;   0-2  N-glycine; 
O-OSN-NaOH. 

0 

1860 

_ 

0 

900 

_ 

6 

1692 

6-9 

6 

829 

5-9 

12 

1545 

6-7 

12 

767 

5-8 

20 

1376 

6-55 

30 

1210 

6-2 

It  is  here,  of  course,  essential  that  the  relation  between  the 
NaOH  and  the  acid  present  (dipeptide+amino-acid)  is  not 
appreciably  changed  by  the  addition  of  glycine. 

Special  mention  must  further  be  made  of  the  work  of 
Abderhalden  and  Gigon  (H.,  1907,  53,  251)  on  the 
hydrolysis  of  glycyl-Z-tyrosine.  In  this  case  the  tyrosine  in  the 
solution  produces  a  considerable  retardation.  Similar  effects 
are  produced  by  the  previous  addition  of  active  amino-acids, 
especially  of  those  occurring  in  nature :  d-alanine,  Z-serine,  Meucine, 
d-glutaminic  acid,  Z-phenylalanine,  d-tryptophane  and  Z-diamino- 
trihydroxydodecanic  acid. 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       143 


The  order  of  magnitude  of  the  retardations  is  shown  by 
the  following  results : 


0-1  grm.  glycyl-Z-tyrosine  + 
1  c.c.  pressed  yeast-juice. 

0-1  grm.  glycyW-tyrosine+1  c  c.  pressed  yeast-  juice 
+  0-05  grm.  d-glutaminic  acid. 

Rotation  corrected 

Minutes. 

Rotation. 

Minutes. 

Rotation. 

for  that  of  the 

glutaminic  acid. 

0 

+0-70° 

0 

+0-70° 

+0-55° 

9 

+0-51 

7 

+0-55 

+0-47 

23 

+0-20 

19 

+0-54 

+0-46 

34 

+0-00 

29 

+0-54 

+0-46 

49 

+0-53 

+0-45 

79 

+0-53 

+0-45 

105 

+0-47 

4-0-39 

On  the  other  hand,  glycine,  Z-alanine  and  d-leucine  exert 
no  retarding  action,  whilst  with  the  racemic  compounds,  such  as 
dZ-alanine,  the  effects  are  small.  From  the  specific  influences 
which  the  hydrolytic  products  thus  show  towards  the  digestion 
of  polypeptides,  it  must  again  be  concluded  that  the  hydrolysing 
enzyme  enters  into  direct  union  with  these  protein  decomposi- 
tion products.  At  about  the  same  time  C  h  o  d  a  t  (Arch. 
Sci.  phys.  nat.,  1907,  26,  112)  made  similar  measurements  with 
the  anhydrides  of  Z-tyrosine  and  glycyl-Z-tyrosine.  In  extremely 
high  dilutions,  the  amino-acids  have  an  accelerating 
action.  Abderhalden  and  G  i  g  o  n  also  point  out  the 
difference  between  digestion  in  vitro,  where  the  decomposi- 
tion products  of  the  proteins  have  a  considerable  retarding 
influence,  and  digestion  in  the  intestinal  canal,  where  these  inhibit- 
ing products  are  rapidly  removed  by  resorption. 

In  other  words:  In  the  organism  we  have  (in  certain  periods) 
so-called  stationary  states,  in  which  the  substance,  acted  on  by 
the  enzyme  and  then  removed  from  the  sphere  of  action  of  the 
latter  by  diffusion  or  some  other  method,  is  continually  replaced 
by  an  equivalent  quantity  of  fresh  starting  material.  Such  cases 
of  stationary  chemical  processes,  for  example,  with  the  unimolec- 
ular  reaction  A— +B+C,  can  be  represented  by  the  following 
scheme. 


Entry  of  A :     *>  « 

C  o 

— »  x  grm.-mols.  !|«2 
per  unit  of  time. «  « 


Field  of  reaction: 
conversion  of  x 


II 


grm.-mols.  per  unit  time.  ~  « 


Exit  of  B  and  C: 

x  grm.-mols.  of 
each  per  unit  time. 


144  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

For  the  given  conditions,  we  have  the  simple  relation : 

dx 

-j-  =  const. 

at 

For  such  a  stationary  condition  to  obtain,  it  is  by  no  means 
necessary  that  a  system  should  be  limited  by  solid  walls.  H. 
Goldschmidt  (Zeitschr.  f.  physikal.  Chem.,  1899,  31,  235) 
has  studied  a  chemical  process  in  which  the  above  equation  is 
realised. 

If  an  excess  of  an  ester,  slightly  soluble  in  water,  is  shaken 
with  a  dilute  hydrochloric  acid  solution,  in  which  the  ester  (in 
so  far  as  it  is  dissolved)  is  hydrolysed  with  a  certain  velocity, 
the  concentration  of  the  dissolved  ester  is  kept  constant  merely 
by  the  shaking,  since  the  amounts  destroyed  by  hydrolysis  are 
continually  removed  by  the  diffusion  of  the  solution.  If  the 
velocity  of  reaction  is  not  very  great,  the  ester  disappearing  from 
the  aqueous  phase  owing  to  the  reaction,  can  be  carried  away 
.completely  by  diffusion,  so  that  the  concentration  of  the  ester 
in  the  aqueous  solution  is  maintained  at  a  constant  value,  namely, 
that  corresponding  with  saturation. 

The  usual  equation  for  a  unimolecular  reaction, 

—  =  (a-x}k 

becomes 

dx  _ ,  „ 

since 

a — x  =  a  =  const. 

A  condition  for  such  a  reaction  is,  therefore,  that  the  phase 
in  which  the  reaction  occurs,  is  always  saturated  with  reference 
to  the  substrate;  this  condition  may  be  fulfilled  by  vigorous 
shaking  of  the  heterogeneous  system,  or  by  employing  the  body 
to  be  dissolved  in  an  extremely  finely  divided  state,  so  that  its 
surface  of  contact  with  the  solution  is  very  great.  Excessively 
laige  surfaces  of  this  kind  occur  particularly  in  so-called  colloidal 
solutions. 


CHEMICAL  DYNAMICS  OF  ENZYME  REACTIONS       145 

In  this  connection,  it  must  be  again  pointed  out  that  the 
acceleration  or  retardation  suffered  by  an  enzyme  reaction  is 
by  no  means  always  to  be  attributed  to  combination  of  the 
enzyme  with  the  substrate.  In  many,  perhaps  even  in  most, 
cases  it  is  a  question  of  the  alteration  of  the  concentration  of  the 
activators  (co-enzyme,  etc.),  either  by  the  reaction  products  and 
the  substrate  acting  in  a  reversible  manner  on  these  substances 
or  by  the  latter  being  changed  or  destroyed  by  secondary  reactions. 
The  opportunity  has  already  been  taken  of  pointing  out  that  the 
deviation  from  the  theoretical  course  of  the  reaction — which  is 
termed  S  c  h  ii  t  z  's  rule — might,  according  to  the  data  as  yet 
to  hand  concerning  peptic  digestion,  very  well  be  caused  by  the 
gradual  fixing  of  the  hydrochloric  acid  by  the  albumoses  and 
peptones  formed,  the  acid  being  withdrawn  from  both  the  sub- 
strate and  the  pepsin.  It  is  to  be  hoped  that  digestion  exper- 
iments carried  out  in  vitro  and  with  a  constant  concentration 
of  hydrochloric  acid,  may  decide  this  not  unimportant  question. 

According  to  theory,  all  reactions,  even  if  they  apparently 
proceed  to  completion,  are  finally  arrested  at  a  position  of  equilib- 
rium. Chemical  reactions  often  go  so  far  in  one  direction  that, 
under  ordinary  conditions,  the  equilibrium  cannot  be  detected 
analytically;  only  in  very  concentrated  solutions  is  the  equilib- 
rium apparent.  The  manner  in  which  the  equilibrium  of  a  rever- 
sible reaction  A^2B  depends  on  the  velocities  of  the  two  opposed 
processes  A— »2B  and  A<— 2B,  and  the  way  in  which  natural  equilib- 
ria and  the  final  states  of  enzymic  reactions  are  connected,  will 
be  described  in  Chapter  VI. 

This  short  resume  of  the  theory  of  enzymic  dynamics  may 
now  be  brought  to  an  end.  It  will  be  seen  later  how  the  exper- 
imental data  agree  with  the  requirements  of  theory. 


146 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


H.    EXPERIMENTAL  DATA  ON  THE  COURSE  OF 
ENZYME  REACTIONS 

ESTERASES  AND  LIPASES 

With  esterases,  that  is,  with  enzymes  which  hydrolyse  lower 
esters  but  not  neutral  fats,  K  a  s  1 1  e  and  Loevenhart 
(Amer.Chem.  Journ.,  1900,  24,  491)  and  Kastle,  Johnston 
and  E  1  v  o  v  e  (ibid,  1904,  31,  521)  have  carried  out  a  number 
of  experiments;  they  used  turbid  aqueous  extracts  of  pig's 
liver  and  pancreas  filtered  through  cloth. 

Tubes  containing  4  c.c.  water,  0-1  c.c.  toluene,  and  0-26  c.c. 
ethyl  butyrate  were  heated  for  5  minutes  at  40°.  One  c.c.  of  a 
10%  extract  was  then  added  and  after  a  definite  time  the  solu- 
tion titrated  with  N/20-KOH  solution: 


Minutes. 

X 

k.  ID* 

K.E 

5 

(6-53) 

1354 

(0-45) 

10 

(8-66) 

907 

(0-40) 

15 

8-53 

597 

0-26 

20 

9-54 

500 

0-24 

25 

10-67 

500 

0-24 

30 

(9-41) 

329 

(0-16) 

60 

17-32 

316 

0-28 

120 

25-35 

244 

0-32 

180 

28-36 

184 

0-28 

The  constant  k,  calculated  from  the  equation  of  a  reaction 
of  the  first  order,  diminishes  regularly  and  very  considerably. 
Also  interpolation  shows  that  the  errors  of  observation,  pre- 
sumably owing  to  the  difficulty  of  pipetting  small  volumes  of  the 
ester  exactly,  were  very  large;  the  values  given  in  brackets  fall 
quite  away  from  the  curve.  For  the  other  numbers,  the  values 
of  K.E  calculated  from  formula  (18) 


a  In 


a— x 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS        147 

show  satisfactory  agreement.  This  formula  was  deduced  (pp. 
133-135)  on  the  assumption  that  the  concentration  of  the  free 
enzyme  is  inversely  proportional  to  that  of  the  products  of  the 
reaction.  The  same  formula  is  obtained  on  the  supposition — 
highly  probable  in  this  case  at  least — that  the  activity 
and  not  the  concentration  of  the  enzyme  is  inversely 
proportional  to  the  reaction  products,  chief  among  these  being 
the  acid.  As  was  found  by  K  a  s  1 1  e  and  Loevenhart, 
the  lipase  of  the  pancreas  is  very  sensitive  to  acids.  It  would 
doubtless  be  of  value  to  ascertain  if  the  formula  for  unimolec- 
ular  reactions  does  not  hold  with  good  approximation  if  a  cer- 
tain quantity  of  strong  acid  were  previously  added  in  order  to 
depress  the  action  of  the  products  of  the  reaction. 

With  a  moderate  degree  of  accuracy  the_  above  figures  and 
those  given  below  agree  with  the  formula  x/\/t  =  k. 

The  measurements  made  by  S  t  a  d  e  on  an  emulsion  of  egg- 
yolk  and  neutralised  gastric  juice  (Hofm.  Beitr.,  1902,  3,  291) 
have  been  calculated  according  to  the  above  formula  by 
Arrhenius  and  found  to  agree  closely  with  it,  as  is  seen  from 
the  following  table : 


Hours. 

x  (observed). 

x  (calculated). 

2 

0-204 

0-186 

K#  =  10 

4 

0-256 

0-257 

6 

0-298 

0-308 

8 

0-353 

0-348 

10 

0-376 

0-383 

25 

0-495 

0-552 

29 

0-515 

0-582 

31 

0-554 

0-596 

35 

0-609 

0-620 

75 

0-775 

0-784 

Further,  Engel  (Hofm.  Beitr.,  1905,  7,  77),  in  a  careful 
investigation  with  emulsion  of  egg-yolk  and  pancreatin  on  the 
lines  of  Volhard's  and  S  t  a  d  e  '  s  experiments,  arrived 
at  the  result  that  S  c  h  u  t  z  's  rule  holds  for  the  enzymic  saponifica- 
tion  of  fats ;  that  is,  for  a  constant  period  of  digestion,  the  amounts 
of  digestion  are  in  the  ratio  of  the  square  roots  of  the  quantities 
of  enzyme,  and  for  the  same  quantity  of  enzyme  the  products 


148 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


of  digestion  are  proportional  to  the  square  roots  of  the  times  of 
digestion.    Hence,  for  one  and  the  same  juice,  the  equation 


must  hold.     This  is  found  to  be  the  case. 


Pancreatin. 

4  hours. 

9  hours. 

25  hours. 

x  (obs.) 

x  (calc.) 

VEi 

x  (obs.) 

x  (calc.) 

X 

V~Ei 

x  (obs.) 

x  (calc.) 

X 

VTsi 

0-04grm. 
0-09    " 
0-16     " 

17-6 
20-9 
35-2 

16-8 
24-5 
31-6 

4-4 
3-5 
4-4 

18-4 
36-3 

48-4 

24-5 
35-0 
44-6 

3-1 
4-0 
4-0 

35-0 
58-2 
72-1 

38-3 
53-0 
65-0 

3-5  15s 
3-8L1 
3.6(| 

The  values  of  x  (calc.)  have  been  obtained  by  means  of  A  r  r- 
h  e  n  i  u  s  's  formula,  the  constant  K  being  taken  as  1.  Although 
the  experimental  errors  are  considerable,  it  is  clear  that  the 
numbers  follow  S  c  h  ii  t  z  '  s  rule. 

The  experimental  data  obtained  by  Z  e  1 1  n  e  r  (Monatsh. 
f .  Chemie,  1905,  26,  727)  with  lipase  from  fly  agaric  (A  m  a  n  i  t  a 
m  u  s  c  a  r  i  a)  have  been  subjected  to  calculation  by  K  a  n  i  t  z  , 
who  found  that  the  results  vary,  as  in  two  series  of  experiments 

T* 

the  quotient  y  was  constant  and  in  another  series  the  expression 
x 

vl- 

E  u  1  e  r  (Hofm.  Beitr.,  1905,  7, 1)  hydrolysed ethylbutyrate in 
aqueous  solution  with  esterases  (from  the  fatty  tissues  of  the 
pig)  and  found  the  law  for  unimolecular  reactions  to  hold. 


Minutes. 
t 

Vol.  of  baryta  solution 
used  in  the  titration 

(C.C.). 

x 

Vol.  of  baryta  solution 
not  used. 

a  —  x 

Unimolecular  reaction 
constant. 

fc.10* 

0 

0 

2-70 



2 

0-3 

2-40 

256 

6 

0-75 

1-95 

235 

9 

1-05 

1-65 

237 

16 

1-65 

1-05 

250 

From    his    observations    on    the    lipase    of    Lactarius 
sanguifluus,     Rouge     (Centralbl.    f.    Bakt.,    1907,    II, 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS        149 


18,  403,  587)  also  drew  the  conclusion  that,  in  dilute  solutions, 
the  action  of  the  enzyme  is  directly  proportional  to  its  amount. 
Experiments  with  the  true  lipases  all  relate  to  systems  with 
limiting  surfaces  perceptible  macroscopically  (suspensions),  since 
most  of  the  lipases  are  insoluble  in  water.  Glycerine  extracts 
showing  lipolytic  activity  can  be  obtained  from  the  pancreatic 
glands  of  the  pig  (A.  K  a  n  i  t  z  ,  H.,  1905,  46,  482).  This  author 
followed  the  course  of  hydrolysis  of  olive  and  castor  oils. 

Into  each  of  a  series  of  test-tubes  were  placed  10  c.c.  of  olive  oil, 
3-9  c.c.  0-lN-sodium  hydroxide,  0-25  c.c.N-calcium  chloride,  and  1  c.c. 
of  lipase  extract,  the  contents  of  the  tubes  being  titrated  after  t  minutes. 
The  numbers  of  c.c.  of  0-lN-sodium  hydroxide  used  are  given  under  x. 


X 

X 

t 

X 

t 

vT 

0 

0-0 



_ 

70 

9-2 

0-131 

1-10 

140 

12-3 

0-088 

1-04 

288 

19-0 

0-065 

1-12 

405 

23-1 

0-057 

1-15 

1455 

34-2 

0-023 

0-90 

The  time-law  of  the  vegetable  lipases  was  first  investigated 
by  Connstein,  Hoyer  and  Wartenberg  (Chem. 
Ber.,  1903,  35,  3988),  who  established  the  essential  fact  that 
considerable  quantities  of  free  acid  are  necessary  for  the  lipases  to 
exhibit  their  action.  If,  as  suggested  by  S  i  g  m  u  n  d  (Monatsh. 
f.  Chemie,  1890,  11,  272),  powdered  castor-oil  seeds  are  ground 
with  water  and  left  for  24  hours  at  about  40°,  small  quantities 
of  acid  are  detectable  by  titration.  Later,  however,  the  quan- 
tity of  acid  formed  rises  suddenly.  This  "  jump  "  occurs  after 
2-3  days  at  35-40°  or  after  4-6  days  at  15-20°. 

For  instance,  5  grms.  of  castor-oil  seeds  were  pounded  with 
5  grms.  of  1%  chloral  hydrate  solution  and  the  mass  kept  at  16°. 


Immediately.    After  2  days. 

Per  cent  of  ricinoleic  acid  found   1  3 


4  days.     6  days.      8  days. 

52         59          59 


This  is  a  case  of  autocatalysis,  in  which  one  of  the  products 
of  the  reaction  exercises  an  accelerating  action.     As  to  the  most 


150 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


favourable  concentration  of  acid,  information  is  given  by  the 
following  results,  obtained  with  sulphuric  acid: 

Normality  of  the  acid 0-02    0-05     0-10    0-12    0-2    0-5 

Percentage  hydrolysed  in  18  hours ...     25        80        86        84        86       13 

In  addition  to  the  seed-lipases,  only  pepsin  acts  in  such  strongly 
acid  solutions  .  According  to  the  authors  named  above,  the  idea 
that  the  acid  transforms  a  pro-enzyme  (zymogen)  present  in  the 
seeds  into  an  active  enzyme,  is  untenable,  since  when  the  seeds 
are  treated  for  a  long  time  with  acid  and  the  latter  then  removed, 
they  exhibit  no  alteration;  in  fact  they  show  as  little  fat-splitting 
action  in  neutral  solution  as  before  treatment,  and  they  also 
become  active  in  presence  of  acid. 

In  their  examination  of  the  influence  of  various  organic  acids 
on  Ricinus-lipase,  H.  E.  Armstrong  and  O  r  m  e  r  o  d  (Proc. 
Roy.  Soc.,  1906,  78,  378)  obtained  the  following  numbers: 


Concentration  of  the  acid. 

0-01  N. 

0-02  N. 

0-10  N. 

0-50  N. 

k.  10*. 

Acetic  acid 

5-45 

14-9 

14-6 

13-6 

1-8 

Succinic  acid      

2-80 

14-6 

15-4 

12-2 

6 

Citric  acid 

7-25 

15-3 

14-7 

1-1 

82 

Tartaric  acid 

6-95 

15-4 

14-2 

97 

Here  also  there  occurs  a  rather  flat  maximum.  No  relation 
is  evident  between  the  strength  of  the  acid — the  dissociation 
constants  are  given  in  the  last  column — and  the  extent  to  which 
the  reaction  is  accelerated  or  the  enzyme  activated. 

The  nature  of  the  added  acid  seems,  indeed,  to  have  but 
slight  influence,  and  this  suggests  the  idea  that  the  action  consists 
of  a  liberation  of  the  true  activator,  itself  presumably  a  weak 
acid. 

In  order  to  ascertain  the  influence  of  the  quantity  of  the 
enzyme  on  the  velocity  of  hydrolysis,  quantities  of  0-5  grm.  of 
the  Ricinus-seeds  were  mixed  well  with  5,  10,  15,  20,  25,  and  50 
grms.  respectively  of  castor  oil  and  with  similar  amounts  of  2% 
acetic  acid.  Assuming  that  the  active  mass  of  the  lipase  is 
proportional  to  the  total  quantity,  and  calculating  the  results 
obtained  after  a  certain  time  by  means  of  the  formula  previously 
deduced  (p.  136) : 

1000 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS        151 


the  following  numbers  are  obtained,  these  agreeing  satisfactorily 
with  the  observed  values  excepting  in  the  case  of  the  largest 
proportions  of  enzyme: 


Action  of  0-5  grm. 

castor-oil 

seeds  on 

After  1  day, 

After  2  days, 

grms.  of  oil.            •  ' 

Solution. 

x  (obs.) 

x  (calc.) 

x  (obs.) 

x  (calc.) 

50 

50 

49 

49 

49 

59 

25 

25 

60 

65 

74 

74 

20 

20 

71 

69 

80 

78 

15 

15 

77 

75 

87 

84 

10 

10 

81 

83 

86 

91 

5 

5 

89 

94 

92 

98 

K  = 

186 

K 

=  300 

The  time-course  of  the  reaction  is  shown  by  a  number  of  the 
experiments  carried  out  by  these  authors,  among  them  Nos. 
26,  28,  38,  and  46.  The  last  two  were  carried  out  by  pounding 
5  grms.  of  castor-oil  seeds  with  6  •  5  grms.  of  castor-oil  and  either 
4  grms.  of  0  •  1  N-sulphuric  acid  (No.  46)  or  4  grms.  of  acetic  acid 
(No.  38).  The  results  were  as  follows: 


EXPERIMENT  46. 


EXPERIMENT  38. 


0-10  N-sulphuric  acid. 

0-10  N-acetic  acid. 

0-40  N-acetic  acid. 

t  (mins.) 

x  (obs.) 

x  (calc.) 

t  (hours) 

*  (obs.) 

x  (calc.) 

t  (hours) 

x  (obs.) 

x  (calc.) 

15 

12 

20 

1 

52 

48-6 

1 

65 

63-9 

30 

20 

27 

2 

65 

62-8 

2 

86 

78-8 

45 

30 

32 

3 

70 

71-5 

3 

84 

86-5 

00 

33 

36 

4 

72 

77-6 

4 

84 

91-2 

90 

41 

43 

24 

80 

99-5 

24 

91 

99-9 

150 

54 

53 

*E=  180 

K.i£  =  380 

210 

59 

59 

330 

68 

69 

1620 

81 

97 

With  the  longer  times,  the  deviations  of  the  observed  numbers 
from  the  calculated  ones  are  considerable  and  on  this  account 
Arrhenius  supposes  equilibrium  to  be  set  up  in  these  cases. 
N  i  c  1  o  u  x  (Soc.  Biol.,  1902,  54,  840)  has  also  made  a  thorough 
investigation  of  Ricinus-lipase  or,  as  he  terms  it,  the  lipolytic 
action  of  the  cytoplasm  of  Ricinus-seeds.  N  i  c  1  o  u  x  like- 


152  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

wise  found  his  preparation  to  be  insoluble  in  water;  he  emulsified 
the  cytoplasm  in  the  oil  —  mostly  cottonseed  oil  —  to  be  hydro- 
lysed  and  added  dilute  acetic  acid.  The  following  numbers  were 
obtained  at  18°: 

t  (minutes)  .......  30        45        60        90       127      150      210       450 

Percent,  hydrolysed  23-6    33-1     40-4     54-8    67-0    73-2    85-5    94-4 


KE  =  lOOOZn—        --  x   1-10    1-58    1-89     2-73    3-45    3-89    5-12    4-31 

1000  -x 
100  100 

k=—  -  log  -          0-388  0-387  0-375  0-382  0-392  0-381  0-399  0.278 
t  100-x 

It  is  evident  that  the  observed  numbers  do  not  follow 
S  c  h  u  t  z  's  rule;  on  the  contrary,  the  course  of  the  hydrolysis 
agrees  moderately  well  with  the  formula  for  unimolecular  reactions. 

A  thorough  series  of  experiments  with  Ricinus-lipase  has 
recently  been  carried  out  by  Jalander  (Biochem.  Z.,  1911,  36,  435). 
For  short  times  (up  to  60  minutes)  be  found  proportionality  with 
enzyme-concentrations  of  1-4  per  1000.  For  more  protracted 
action  this  proportionality  disappears,  S  c  h  ii  t  z  '  s  rule,  x:VE  = 
const,  then  holding  approximately. 

Bodenstein  and  Dietz  (Zeitschr.  f.  Elektrochem., 
1906,  12,605;  Dietz,  H.,  1907,  52,  279)  have  also  studied  a 
heterogeneous  system.  Pancreatic  lipase,  in  the  shape  of  shavings 
of  the  tissue  of  the  pancreatic  glands  of  the  pig,  was  emulsified 
with  amyl  alcohol  containing  water  and  butyric  acid  or  amyl 
butyrate  in  solution. 

One  would  expect  the  velocity  equation, 

~TT  =  ^1  *  ^  acid  "  C»  alcohol       K>2  '  C  ester  '  C  water 

at 

to  be  fulfilled,  all  the  concentrations  relating  to  the  enzyme 
phase.  The  concentrations  of  water  and  alcohol  are  approx- 
imately constant,  since  only  very  small  quantities  of  these  dis- 
appear during  the  process.  On  the  other  hand,  the  substances 
must  be  divided  between  enzyme  and  liquid  according  to 
Nernst's  law  of  distribution,  i.e.,  Cenzyme  =  a.  Csoiution.  The 
proportionality  factors,  like  the  constant  concentrations  of 
alcohol  and  water,  become  included  in  the  constants,  so  that 

--  =  ki  •  Cacld  —  &2  •  Cester  =  &1  (a  —  x)  —  k2X. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       153 

If  but  little  water  is  dissolved  in  the  amyl  alcohol,  the  reverse 
reaction  is  very  slight  and  the  process  goes  on  according  to  the 
simple  equation:. 

dx     , 


as  is  shown  by  the  following  table : 

a  =  0  •  10  normal 


4%  H20. 

2%  H2O. 

t  (hours). 

Titre  of  5  c.c. 

k 

t  (hours). 

Titre  of  5  c.c. 

k 

0-00 

13-55 



0-00 

13-55 

- 

2-00 

12-75 

0-013 

2-50 

13-15 

0-0064 

5-53 

11-90 

0-010 

9-57 

11-80 

0-0062 

10-33 

10-35 

0-012 

14-35 

10-95 

0-0064 

15-17 

9-15 

0-012 

24-45 

9-45 

0-0063 

25-20 

7-05 

0-012 

31-88 

8-45 

0-0063 

32-58 

5-85 

0-012 

47-89 

6-55 

0-0065 

48-83 

4-25 

0-012 

55-27 

5-90 

0-0064 

55-92 

3-90 

0-011 

76-67 

4-60 

0-0060 

77-73 

3-00 

0-010 

100-42 

3-25 

0-0062 

308-00 

1-50 

— 

308-00 

1-10 

— 

On  passing  to  higher  contents  of  water,  a  final  condition 
attainable  from  either  side  is  set  up  and  the  velocities  of  the  two 
opposed  reactions  become  measureable.  Such  a  case  is  presented 
by  the  following  tables. 


Formation  of  ester. 


Hydrolysis  of  ester. 


t  (hours). 

Titre  of  5  c.c. 

ki 

t  (hours). 

Titre  of  5  c.c. 

h 

0-00 

13-40 

— 

0-00 

0-00 



1-98 

12-60 

0-014 

3-63 

0-75 

0-0075 

4-00 

11-82 

0-014 

7-60 

1-45 

0-0077 

6-98 

10-80 

0-014 

16-77 

2-35 

0-0064 

11-55 

9-10 

0-016 

24-05 

2-95 

0-0070 

14-98 

8-40 

0-016 

89-30 

4-20 

— 

25-10 

6-55 

0-016 

96-95 

4-25 

— 

Mean  0-015 

Mean  0-0072 

154 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Both  constants  are  approximately  doubled  if  the  quantity 
of  enzyme  is  doubled: 

Quantity  of  enzyme.  ^    Constants^ 

1 0-015          0-0072 

2 0-028          0-014 

A  .  E  .  Taylor,  who  hydrolysed  triacetin,  the  acetic  ester 
of  glycerine,  with  powdered  Ricinus-seeds,  found  the  course  of 
the  reaction  to  be  as  with  unimolecular  reactions.  He  gives  the 
following  results  for  experiments  in  which  0-5,  1,  and  2%  solu- 
tions of  triacetin  were  employed.  The  constants  k  refer  to  18°. 


t  (hours). 

4 

8 

16 

24 

28 

32 

40 

48 

0-5% 

x  (obs.) 

0-096 

0-162 

0-287 

0-418 

0-489 

0-477 

0-623 

0-652 

k 

109 

96 

92 

98 

104 

88 

106 

96 

1-0 

x  (obs.) 

0-083 

0-174 

0-338 

0-418 

0-488 

0-542 

0-609 

0-655 

k 

94 

104 

112 

98 

104 

106 

102 

96 

2-0 

x  (obs.) 

0-098 

0-174 

0-323 

0-431 

0-502 

0-485 

0-595 

0-636 

k 

112 

104 

106 

102 

108 

90 

98 

91 

How  it  happens  that  in  all  these  experiments  the  quantity  of 
fat  hydrolysed  can  be  calculated  by  means  of  the  very  simple 
law  holding  for  homogeneous"  systems,  is  not  very  easy  to  under- 
stand. With  the  hydrolysis  of  fats  and  of  triacetin  this  is  all 
the  more  remarkable,  since  this  hydrolysis  takes  place  in  three 
stages,  which  doubtless  occur  with  different  velocities. 

New  experiments  with  esterase  from  pig's  liver  have  been  made 
by  G.  Peirce  (Journ.  Amer.  Chem.  Soc.,  1910,  32,  1517). 
The  principal  results  are  as  follows: 

(1)  In  a  solution  of  given  volume  and  given  acidity,  the  time 
required  for  the  hydrolysis  of  a  definite  quantity  of  ethyl  butyrate 
is  inversely  proportional  to  the  concentration  of  the  enzyme. 
Under  similar   conditions  of  acidity,   each  particle  of  enzyme 
hydrolyses  the  same  absolute  amount  of  ester  per  instant,  no 
matter  what  the  concentration  of  the  enzyme. 

(2)  With  a  given  concentration  of  enzyme,  the  time  taken 
to  hydrolyse  a  given  amount  of  ethyl  butyrate  is  dependent  on 
the  acid-concentration  but  not  on  the  ester-concentration,  pro- 


EXPEKIMENTAL  DATA  OF  ENZYME  REACTIONS       155 

vided  this  is  above  N/200.  In  other  words,  for  each  concentra- 
tion of  acid  a  given  amount  of  enzyme  hydrolyses  very  nearly  the 
same  amount  of  ethyl  butyrate  over  a  wide  range  of  ester-con- 
centration. 

(3)  This  phenomenon  can  be  brought  into  conformity  with 
the  law  of  mass  action  by  assuming  that  the  enzyme  and  the  ester 
form  an  intermediate  compound,  which,  in  concentrations  of 
the  ester  above  N/200  contains  most  of  the  enzyme. 


AMYLASE 

The  chemical  process  of  the  hydrolysis  of  starch  presumably 
takes  place  in  several  phases,  in  which  the  dextrins  formed  as 
intermediate  products  are  broken  down;  amylase  may  con- 
sequently be  composed  of  several  enzymes. 

In  experiments  with  diastase  (amylase),  the  difficulties 
attending  the  varying  constitution  of  the  enzyme  are  supplemented 
by  the  complication  introduced  by  a  non-individual  substrate. 
As  has  been  shown,  more  especially  by  Maquenne's  inves- 
tigations, starch  consists  in  reality  of  two  components,  namely, 
80-85%  of  amylose  and  15-20%  of  amylopectin.  A  m  y  1  o  s  e 
forms  no  paste  and  in  solution  is  coloured  an  intense  blue  by 
iodine;  only  in  solution  is  it  attacked  by  malt-diastase.  There 
exist  a  series  of  condensation  products  increasingly  soluble  in 
water,  amylose  being  the  last  of  these;  the  lower  members  more 
especially  are  largely  contained  in  "  soluble  starch."  In  the 
saccharification  of  pure  amylose  maltose  alone  is  formed.  Amylo- 
pectin is  a  gelatinous  substance  which  is  insoluble  in  water  and 
alkalis  and  gives  dextrin  as  well  as  maltose  (?)  on  hydrolysis. 
It  is  not  yet  certain,  but  is  probable  that  this  is  brought  about 
by  a  special  amylopectinase  and  not  by. the  amylase.  In  any 
case,  in  quantitative  experiments  on  amylase  the  purest  possible 
amylose  or,  at  any  rate,  "  soluble  starch  "  should  be  employed. 
For  the  technical  determination  of  diastase,  potato  starch  is 
treated  according  to  L  i  n  t  n  e  r  's  method  (see  Appendix). 

As  regards  the  hydrolysis  curves,  the  experiments  made  by 
different  authors  do  not  agree  especially  well.  The  earliest  of  these 
experiments,  carried  out  by  H  .  Brown  and  G  1  e  n  d  i  n  - 
n  i  n  g  (Journ.  Chem.  Soc.,  1902,  81,  388)  with  malt-extract  do 


156 


GENERAL  CHEMISTRY   OF  THE  ENZYMES 


not  correspond  with  the  simple  logarithmic  curve. 
are  expressed  better  by  the  formula 

1  ,      a+x 


The  results 


as  is  shown  by  the  following  numbers. 

3%  solution  of  starch  with  0-25  c.c.  malt-extract  per  100  c.c.: 


Minutes. 

X 

fc.105 

fctf.105 

Temperature,  51-52°. 

10 

0-1084 

498 

'472 

20 

0-2250 

553 

497 

40 

0-4350 

620 

506 

60 

0-6150 

690 

518 

80 

0-7385 

728 

514 

100 

0-8150 

732 

495 

120 

0-8800 

762 

497 

140 

0-9220 

791 

497 

160 

0-9500 

813 

492 

3%  solution  of  starch  with  1  c.c.  malt-extract  per  100  c.c. 


10 

0-081 

366 

352 

Temperature,  21° 

20 

0-163 

386 

357 

40 

0-308 

399 

345 

60 

0-440 

419 

341 

70 

0-506 

437 

345 

Henri's  experiments  (Lois  generates  etc.),  on  the  other 
hand,  follow  the  logarithmic  law;  whether  the  method  employed 
(determination  of  the  change  of  reducing  power)  is  free  from 
objection  depends  on  the  purity  of  the  amylase  used. 

Also  with  saliva-diastase,  A  .  E  .  Taylor  obtained  the 
following  values: 


Substrate  0-25% 

t  (minutes.) 

30 

45 

60 

75 

90 

120 

150 

180 

fc(X106) 

490 

465 

455 

470 

465 

455 

460 

455 

Substrate  0-5% 

k  (X106) 

430 

420 

390 

415 

405 

395 

430 

410 

Substrate  0-75% 

k  (X106) 

390 

370 

385 

390 

380 

370 

365 

370 

As  Taylor  emphasised,  the  values  of  k  vary  considerably 
with  the  concentration  of  the  substrate. 

In  the  course  of  a  valuable  investigation  on  pancreas-amylase, 
Kendall  and  Sherman  (Journ.  Amer.  Chem.  Soc.,  1910, 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       157 


32,  1087)  found:  (1)  that  the  initial  speed  of  conversion  for  a 
constant  amount  of  enzyme  was  the  same  for  different  concentra- 
tions of  starch;  (2)  that  the  speed  of  the  reaction  diminished 
the  more  rapidly,  the  smaller  the  initial  concentration  of  the  starch. 

For  the  formula  deduced  by  these  investigators  to  express 
the  course  of  the  reaction,  their  original  paper  must  be  consulted. 

As  regards  the  influence  of  the  concentration  of  the  amylase, 
Henri  found,  with  a  vegetable  preparation,  proportionality 
between  the  concentration  of  the  enzyme  and  the  quantity  of 
starch  hydrolysed  per  unit  of  time. 

Taylor  arrived  at  the  same  result  with  saliva-diastase. 
Brown  and  Glendinning  (Journ.  Chem.  Soc.,  1902, 
81,  381),  however,  found  the  velocity  of  reaction  to  be  propor- 
tional to  the  square-root  of  the  concentration  of  the  saliva-diastase, 
and  Klempin  (Biochem.  Z.,  1908,  10,  206),  from  experiments 
with  oat-diastase,  concluded  that  S  c  h  ii  t  z  's  rule,  E^/i=  K, 
holds  for  this  enzyme. 

P  a  w  1  o  w  's  experiments  (Arbeit  der  Verdauungsdrlisen, 
Wiesbaden,  1898),  which  were  carried  out  with  M  e  1 1 's  capillary 
tubes  and  are  of  interest  in  themselves,  hardly  give  any  informa- 
tion concerning  the  course  of  the  saccharification  of  starch,  since 
they  dealt  with  the  liquefaction  of  starch-paste  and  since  also 
the  velocity  of  diffusion  exerts  a  determining  influence  on  the 
velocity  of  the  process. 

A  large  number  of  experiments  were  made  by  Mdlle. 
P  h  i  1  o  c  h  e  (Journ.  de  Chim.  physique,  1908,  6,  213,  355)  with 
"  Merck "  diastase  and  taka-diastase,  starch  and  glycogen 
being  used  as  substrates. 

The  constants  of  the  formula  for  unimolecular  reactions  di- 
minish rapidly  as  the  reaction  proceeds.  The  following  numbers 
were  obtained  with  1%  starch  solution,  the  concentration  of  the 
diastase  being  1 :  20,000. 


Minutes. 

X 

1    a 

a 

t  a—x' 

21 

0-06 

123 

51 

0-11 

100 

113 

0-15 

62 

224 

0-26 

58 

390 

0-36 

50 

158  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

The  influence  of  the  concentration  of  the  starch  on  the  amount 
of  maltose  formed  in  60  minutes  is  shown  by  the  following  table: 

Concentration  of  starch  (%).  Quantity  of  maltose  (60  mins.). 

1  0-24 
1-5  0-30 

2  0-338 
2-5  0-397 

3  0-397 

Activators  and  Inhibitors.  The  following  substances 
favour  the  hydrolysis  of  starch:  vanadium  and  aluminium  salts,  ammo- 
nium and  calcium  phosphates,  asparagine,  ammo-acids,  proteins  and 
picric  acid  (Ef front,  Enzymes  and  their  Applications,  p.  117; 
Soc:  Biol.,  1904,  57,  234;  Allg.  Brauer-  und  Hopfenzeitung,  1905,  45). 
The  accelerations  are  sometimes  very  considerable;  thus  an  addition  of 
0-05  grm.  of  asparagine  to  100  c.c.  of  a  starch  solution  containing 
amylase  increases  the  velocity  sevenfold.  Carbon  dioxide  also  has  an 
accelerating  action,  especially  when  under  increased  pressure  (D  e  t  m  e  r  , 
Mtiller-Thurgau). 

The  optimum  activity  of  vegetable  diastases  occurs  with  a  slight 
excess  of  hydrogen-ions  (small  quantities  of  organic  acids);  hydroxyl- 
ions,  even  in  very  small  concentration,  retard  the  hydrolysis,  but  the 
inactivation  is  annulled  immediately  the  alkali  is  neutralised.  The 
optimum  temperature  (measured  by  K  j  e  1  d  a  h  1  by  the  reducing 
power  of  the  hydrolytic  products)  is  63°. 

INVERTASE 

On  the  hydrolysis  of  cane-sugar  by  invertase  numerous 
quantitative  investigations  have  been  carried  out.  Of  these, 
besides  the  earlier  work  of  K  j  e  1  d  a  h  1  (Medd.  fra.  Carlsberg 
Lab.,  1881),  mention  must  first  be  made  of  the  researches  of 
T  a  m  m  a  n  n  (Zeitschr.  f.  physikal.  Chem.,  1889,  3,  25)  and  of 
those  executed  almost  simultaneously  by  O  '  S  u  1 1  i  v  a  n  and 
T  o  m  p  s  o  n  (Journ.  Chem. Soc.,  1890, 57, 834).  These  investiga- 
tors showed  first  that  the  reaction  is  a  catalytic  one  and  O  '  S  u  1  - 
1  i  v  a  n  and  T  o  m  p  s  o  n  found  it  to  be  unimolecular.  This 
result  was  contested  later  by  D  u  c  1  a  u  x  (Traite  de  Micro- 
biologie,  Vol.  2,  129),  while  Henri  (Zeitschr.  f.  physikal. 
Chem.,  1902,  39,  194)  also  arrived  at  a  formula  differing  from 
the  unimolecular  one,  namely,  the  expression  (compare  p.  129), 

1          a+x 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       159 


which  corresponded  moderately  well  with  his  experimental  data. 
Still  later  Bodenstein  deduced  the  complicated  formula 
mentioned  on  p.  129,  and  finally  Henri  (Theses,  p.  92) 
arrived  at  the  following  equation: 


a   1  .  1  i         a 
—  J  +-f  log  -_-, 


which  he  obtained  by  integration  of  the  differential  Eq.  (12) 
of  p.  131.  As  has  been  already  stated,  it  has  recently  been  found 
that  the  experimental  data  of  Henri  and  his  collaborators 
are  considerably  influenced  by  the  mutarotation  of  glucose 
and  therefore  give  no  definite  information  as  to  the  time-course 
of  the  hydrolysis  of  cane-sugar;  further,  in  these  experiments 
the  concentration  of  the  hydrogen-ions  was  not  defined.  Hud- 
son (Journ.  Amer.  Chem.  Soc.,  1908,  30,  1160,  1564)  has  per- 
formed a  valuable  service  not  only  in  demonstrating  the  reliabil- 
ity of  O  '  S  u  1  1  i  v  a  n  and  Tompson's  data,  but  also  in 
continuing  and  considerably  extending  the  investigations  of 
these  workers. 

O  '  S  u  1  1  i  v  a  n  and  T  o  m  p  s  o  n  had  rightly  recognised  that 
glucose  formed  from  cane-sugar  by  inversion  appears  firstly  in  a 
mutarotating  condition  and  that  therefore  the  optical  activity 
of  a  solution  which  has  been  inverted  by  enzyme  affords  no  measure 
of  the  progress  of  the  reaction.  To  the  test-portions  removed 
after  definite  times  from  their  solutions  they  hence  added  a  little 
alkali,  which  annuls  the  mutarotation  almost  instantaneously. 
Their  results  show  that  experiments  carried  out  in  this  way 
correspond  with  the  unimolecular  formula,  but  that,  if  the  pre- 
caution mentioned  is  not  taken,  the  constant  increases  con- 
siderably. Their  numbers  are  as  follows  : 


Rotation. 

1  .       a 
k  =—ln  . 

x       a—x 

Minutes. 

Without  alkali. 

With  alkali. 

Without  alkali. 

With  alkali. 

0 

74-5° 

69-4° 





37 

57-9 

37-6 

0-0021 

0-0046 

152 

0-6 

-20-4 

0-0037 

0-0074 

268 

-19-1 

-24-8 

0-0041 

0-0058 

00 

-27-8 

-27-8 

— 

— 

160 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


The  constancy  of  the  figures  in  the  final  column  leaves  some- 
thing to  be  desired  and  Hudson's  numbers  may  well  be  given 
here: 


Rotation. 

10«*-^.  log  -^-. 

t            a-x 

Minutes. 

Without  alkali. 

With  alkali. 

^Without  alkali. 

With  alkali. 

0 

24-50° 

24-50° 





30 

16-85 

14-27 

396 

558 

60 

10-95 

7-90 

399 

530 

90 

4-75 

3-00 

464 

539 

110 

1-95 

0-80 

482 

534 

130 

-0-55 

-1-49 

511 

559 

150 

-2-20 

-2-40 

522 

533 

00 

-7-47 

-7-47 

As  is  seen  from  this  and  other  tables  given  by  Hudson, 
the  values  of  k  are  constant. 

According  to  Sorensen  (Biochem.  Z.,  1909,  21,  131) 
and  Michaelis  and  Davidsohn  (Biochem.  Z.,  1911, 
35,  386),  however,  this  constancy  of  the  reaction-coefficient  is 
observed  only  with  a  certain  concentration  of  the  hydrogen-ions. 
With  different  H'-concentrations,  Sorensen  obtained  the 
following  results: 

Temperature  52° 


H'=0-2.10-6 

H'  =  0-1.10-3 

H-=0-2.10~3 

t 

k 

t 

k 

/ 

k 

2 



2 



2 



17 

91 

17 

127 

17 

53-6 

32 

103 

32 

127 

32 

39-3 

47 

111 

47 

132 

47 

26-1 

62 

127 

62 

135 

62 

18-2 

92 

147 

92 

149 

92 

15-3 

122 

230 

122 

126 

122 

11-2 

While,  therefore,  with  H'-concentration  of  0-2.10  6,  the  coefficient 
k  increases,  with  H*  =0- 1.10~3  it  remains  constant 
and  with  H'=0-2.10~3  it  diminishes.  Michaelis  and 
Davidsohn  explain  this  behaviour  on  the  assumption  that 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       161 


the  constancy  of  the  values  of  k  is  brought  about  by  the  com- 
pensating effect  of  the  destruction  of  the  enzyme  on  the  increasing 
values  of  the  coefficient.  Considering  the  high  temperature,  52°, 
chosen  for  Sorensen's  experiments,  this  view  is  most  prob- 
ably accurate,  and  at  lower  temperatures,  and  higher  concentra- 
tions of  hydrogen-ions  (H'  =  10~3)  M  i  c  h  a  e  1  i  s  and  David- 
s  o  h  n  found  increasing  values  for  k. 

That  other  experimenters  have  been  unable  to  obtain  agreement 
with  the  law  of  mass  action,  cannot  therefore,  be  due  solely  to  non- 
removal  of  the  mutarotation.  Thus,  H.  E.  Armstrong  and 
Glover,  in  comparing  the  actions  of  invertase  on  cane-sugar  and 
on  raffinose  (Proc.  Roy.  Soc.,  1908,  80,  312),  rendered  the  sugar  solutions 
alkaline  before  reading  them  in  the  polarimeter,  but  still  obtained  no 
greater  constancy,  as  is  shown  by  the  figures  in  the  left-hand  part  of 
the  following  table: 


34-2  grms.  cane-sugar 
+4  c.c.  invertase-extract  per  1000  c.c. 

59-4  grms.  raffinose 
+4  c.c.  invertase-extract  per  1000  c.c. 

Minutes. 

Percentage 
hydrolysed. 

&.105. 

Percentage 
hydrolysed. 

fc.105. 

0 

0-0 



0-0 



5 

8-3 

753 

1-8 

157 

15 

25-9 

868 

5-9 

176 

25 

39-5 

865 

13-4 

249 

40 

62-4 

1062 

20-1 

243 

60 

78-2 

1102 

29-3 

251 

95 

01-1 

1106 

41-6 

246 

140 

93-7 

859 

53-4 

237 

200 

95-1 

656 

66-6 

238 

260 

96-0 

537 

77-9 

252 

oo 

100-0 

These  tables  show  that  invertase  hydrolyses  cane-sugar  into  fructose 
and  glucose  about  four  times  as  rapidly  as  this  enzyme  decomposes 
raffinose  into  fructose  and  melibiose. 

Our  knowledge  of  the  other  conditions  governing  the  action 
of  invertase  is  due  to  the  work  ofO'Sullivan  and  T  o  m  p  - 
son  and  of  H  u  d  s  o  n,  confirmation  of  which  has  been  supplied 
by  Taylor  (Journ.  of  Biol.  Chem.,  1909,  5,  405).  The  pro- 
portionality found  by  the  first-named  authors  to  exist  between 
the  velocity  of  inversion  and  the  concentration  of  the  enzyme 
is  completely  confirmed  by  H  u  d  s  o  n  's  results. 


162 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


O  '  S  u  1 1  i  v  a  n    and  T  o  m  p  s  o  n  give  the  following  table 
(1  o  c  .  c  i  t .  ,  p.  848) : 


Temp. 

Invertase 
prepara- 

N-H2SO4. 

Time  readings. 

Reading 
in  2  d.m. 

Minutes  taken  to 
reach  zero  rotation. 

tion,  grms. 

tube. 

Beginning. 

End. 

A  (obs.). 

B  (calc.). 

15-5° 

0-15 

0-00187 

11    40 

4  41 

-    2-0° 

283-0 

291 

15-5 

0-45 

0-0031 

3  00 

4  40 

-   1-8 

94-8 

96-3 

15-5 

1-50 

0-0050 

11  56 

12  26 

+  1-0 

30-7 

29-1 

56-5 

0-0345 

0-00025 

11  00 

12  43 

+  16-5 

157-6 

157-1 

56-5 

0-0722 

0-000375 

11  22 

12   15 

+  13-5 

74-8 

75-1 

The  third  column  gives  the  acidity  of  the  solution,  the  con- 
centrations of  acid  used  having  been  found  by  preliminary 
experiments  to  be  the  most  favourable  to  the  velocity  of  reaction. 
In  the  last  column  but  one,  marked  A,  are  given  the  times  elapsing 
before  the  rotation  falls  to  0°  and  in  the  last  column,  B,  the  times 
which  would  be  necessary  for  this  to  occur  on  the  assumption 
of  proportionality  between  concentration  of  enzyme  and  velocity 
of  reaction. 

Also  with  change  of  the  concentration  of  sugar,  the  require- 
ments of  theory  seem  to  be  satisfied  within  very  wide  limits  by 
O'  S  u  1 1  i  v  a  n  and  Tompson's  results,  that  is,  in  equal 
times  one  and  the  same  quantity  of  enzyme  hydrolyses  equal 
proportions  of  the  sugar,  no  matter  what  the  concentration  of  the 
latter  may  be. 

On  the  other  hand,  A  .  J  .  Brown  (Journ.  Chem.  Soc.,  1902,  81, 
373)  gives  numbers  indicating  that  a  given  quantity  of  enzyme  inverts 
the  same  absolute  amount  of  sugar  in  a  definite  time: 


Grms.  of  cane-sugar 
per  100  c.c. 

Grms.  of  cane-sugar  inverted 
in  60  minutes. 

Percentage  of  cane-sugar 
inverted  in  60  minutes. 

4-89 

1-230 

25-2 

9-85 

1-355 

13-8 

19-91 

1-355 

6-8 

29-96 

1-235 

4-1 

40-02 

1-076 

2-7 

But  in  dilute  sugar  solutions  containing  relatively  large  amounts  of 
enzyme,  the  action  of  a  given  quantity  of  enzyme  is,  according  to 
Brown,  proportional  to  the  concentration  of  the  sugar. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       163 


Grms.  of  cane-sugar 
per  100  c.c. 

Grms.  of  cane-sugar 
inverted  in  60  minutes. 

;       lO'fc^log^-. 
t          a—x 

(2-0) 

(0-308) 

(132) 

1-0 

0-249 

219 

0-5 

0-129 

239 

0-25 

0-060 

228 

Hudson  collects  his  results  in  the  following  table : 

Influence     of    the    Concentration    of  Inver- 
tase   on    the  Velocity    of    Inversion  at  30° 


Percentage  of  cane-sugar  inverted  with  the 

Concentration 
of  the 

Minutes. 

Product. 

following  amounts  of  cane-sugar  per  litre. 

invertase, 

E. 

t 

E.t. 

A  c   c  grms. 

90-9^: 
litre 

o_o   grms. 

litre 

litre 

2-00 

15 

30 

73-2 

45-3 

11-2 

2-00 

30 

60 

93-0 

74-2 

22-0 

1-50 

20 

30 

73-2 

44-8 

11-2 

1-50 

40 

60 

92-8 

74-5 

22-7 

1-00 

30 

30 

72-9 

45-3 

11-5 

1-00 

60 

60 

93-0 

74-7 

22-3 

0-50 

60 

30 

72-9 

45-2 

11-4 

0-50 

120 

60 

92-7 

74-5 

22-6 

0-25 

120 

30 

73-1 

45-2 

10-9 

0-25 

240 

60 

92-7 

74-7 

21-9 

The  results  are  therefore  as  follows : 

1.  Proportionality  exists  between  the  amount  of  sugar  inverted 
per  unit  of  time  and  concentration  of  the  enzyme. 

2.  The  concentration  functions  are,  in  addition,  dependent 
on  the  relative    quantities  of  substrate  and  enzyme.     So 
long  as  the  enzyme  is  not  present  in  large  excess  the  relative 
amount  of  hydrolysis  diminishes  as  the  amount  of  substrate  is 
increased. 

With  his  erroneous  method,  Henri  observed  a  relation  indicated 
by  the  following  table.  The  number  of  milligrams  of  sugar  inverted 
after  the  first  minute  in  a  c-normal  cane-sugar  solution  is  denoted  by  n: 


0-58 
0-01 


1-41 
0-025 


2-40 
0-05 


2-96    4-65 
0-10    0-25 


5-04 
0-50 


4-45    2-82 
1-00     1-50 


1-15 
2-00 


Apart  from  the  fact  that  Hudson'  s   investigation  has  led 
again  to  a  method  which  is  free  from  objection,  the  observation 


164 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


that  cane-sugar  gives  rise  first  of  all  to  a-glucose  possesses  on 
little  interest.  (Its  bearing  on  the  enzymic  synthesis  of  disac- 
charides  will  be  considered  later.)  From  the  work  referred  to 
we  may  take  the  following  extract: 

From  cane-sugar,  not  only  glucose  but  also  fructose  is  formed 
in  a  labile  (characterised  by  high  rotatory  power)  modification. 
But  the  disappearance  of  the  mutarotation — measured  at  30° — • 
in  the  case  of  fructose  (constant  of  reaction  k  =  0  •  186)  proceeds 
about  11  times  as  fast  as  with  glucose  (k  =  0-0167).  Dissolved 
invertase-preparations  have  no  influence  on  these  velocity  con- 
stants. Hence,  in  a  cane-sugar  solution  undergoing  enzymic 
hydrolysis,  the  difference  between  the  apparent  and  the  actual 
degree  of  hydrolysis  depends  almost  entirely  on  the  alteration 
of  the  rotation  of  the  glucose.  If  by  means  of  a  very  active 
invertase,  a  cane-sugar  solution  can  be  inverted  almost  instan- 
taneously, all  further  alteration  in  the  rotation  of  the  solution 
must  be  attributed  almost  wholly  to  the  mutarotation  of  the  glu- 
cose and  the  velocity  of  this  change  must  be  nearly  coincident  with 
the  fall  of  rotation  occurring  with  pure  glucose.  This  has  now 
been  actually  confirmed. 

Use  was  made  of  an  invertase  solution  so  active  that  72% 
of  the  cane-sugar  was  inverted  within  half  a  minute.  The  final 
rotation  was  determined  after  addition  of  a  little  sodium  hydroxide 
solution: 


Minutes. 

Rotation  without 
alkali. 

10'  .          a 
-—•log  — 
t            a—x 

Minutes. 

103  ,           a 
log  — 
t            a—x 

0 

33-50° 

— 

— 

— 

3 

11-88 

99 

— 

— 

4 

7-32 

99 

— 

— 

5 

4-77 

93 

— 

— 

9 

-  0-35 

72 

— 

— 

10 

-  1-35 

69 

— 

— 

13 

-  3-57 

63 

0 

Commencement 

16 

-  4-90 

57 

3 

32-3 

19 

-  6-03 

54 

6 

33-4 

23 

-  7-15 

50 

10 

33-6 

29 

-  7-92 

44 

16 

28-7 

30 

-  8-22 

45 

17      . 

30-7 

00 

-10-22 

— 

— 

— 

31  '7 

EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       165 

In  an  equally  concentrated  solution  of  invertase  the  velocity 
constant  of  the  disappearance  of  the  mutarotation  of  glucose 
was  found  to  be  /b.!03  =  29-9,  which  is  in  good  agreement  with 
the  value,  31-7,  given  above. 

Hudson's  excellent  investigations  have  been  treated  at 
length,  because  the  results  obtained  with  invertase  are  of  impor- 
tance to  the  consideration  of  the  enzymic  decomposition  of  other 
disaccharides  and  of  the  glucosides,  where  the  mutarotation  of 
the  hexoses  makes  itself  felt  to  a  greater  or  less  extent  as  a  source 
of  error. 

Worthy  of  notice  is  the  influence  of  the  products  of  the  reac- 
tion on  the  course  of  inversion.  As  is  shown  by  the  experiments 
ofE.F.  Armstrong  (Proc.  Roy.  Soc.,  B,  1904,  73,  500), 
Barendrecht  (Zeitschr.  f.  physikal.  Chem.,  1904,  49,  456) 
and  also  Henri  (loc.  cit.),  fructose  retards  inversion  to  a  much 
greater  extent  than  glucose  does.  According  to  Baren- 
drecht, galactose  also  exercises  a  retarding  influence,  which 
is,  however,  less  than  that  of  fructose. 

The  marked  acceleration  of  enzymic  inversion  by  acids  has 
already  been  mentioned. 

It  was  found  by  O'Sullivan  and  T  o  m  p  s  o  n  that  invertase 
is  very  sensitive  to  small  amounts  of  acid,  and  this  observation  has 
been  confirmed  by  Hudson  (loc.  cit.).  According  to  the  work  of 
the  latter,  invertase  shows  its  optimum  activity  in  about  0  •  0006  normal 
hydrochloric  acid.  Sorensen,  who  made  a  very  thorough  inves- 
tigation of  the  influence  of  acidity  on  enzyme  actions  (Biochem.  Z., 
1909,  21,  131)  found  the  optimum  concentration  of  the  hydrogen-ions 
for  invertase  to  be  W~4  '4  — 10 ~4'6.  Very  weak  acids,  like  carbonic 
acid,  produce  corresponding  accelerations. 

The  slightest  excess  of  OH-ions  brings  the  reaction. to  a  standstill. 

The  enzyme  is  almost  entirely  uninfluenced  by  antiseptics  such  as 
toluene  and  chloroform. 

The  temperature-optimum  is  given  as  50-60°.  The  inactivation 
constant,  &t!03,  of  invertase  from  a  yeast  of  Frohberg  type  was  found 
to  have  the  value  4  at  60°  and  with  a  H'-concentration  of  10  ~6  in 
aqueous  solution  (a  f  U  g  g  1  a  s  ,  H.,  1910,  65,  124). 

The  velocity  of  inversion  by  living  yeast  has  been 
studied  byEuler  andS.Kullberg  (H.,  1911,  71,  24). 

In  this  case  the  system  is  macro-heterogeneous,  so  that  the 
velocity  of  diffusion  should  exert  an  influence  on  the  course  of  the 
process. 


166 


GENERAL   CHEMISTRY  OF  THE  ENZYMES 


Here   also  the  inversion   corresponds  with  the  formula  for 
unimoleeular  reactions. 

Temperature,  20°. 


Inversion  mixture. 

Time 
(mins.). 

Rotation. 

A-x 

&.1O>. 

la 

0-25grm.  fresh  distillery  yeast 
+20  c.c.  20%  cane-sugar  solu- 
tion +5  c.c.  H2O  +  1  c.c.  chlo-  i 
reform;      the     reaction    was 
stopped  by  5  c.c.  0-4N-NaOH 
solution  

0 
17 
25 
34 

00 

7-15° 
6-25 
5-80 
5-25 
-2-29 

9-44 

8-54 
8-09 
7-54 

26 

27 
29 

0 

7-15 

9-44 

— 

16 

Same  as  above,  but  without  ad- 
dition of  chloroform  

17 
25 

6-20 

5-84 

8-49 
8-13 

27 
26 

36 

5-30 

7-59 

26 

00 

-2-29 

— 

— 

It  will  be  seen  that  chloroform  exerts  no  influence  on  inversion 
by  living  yeast.  Between  20  and  30 °,  the  effect  of  the  tem- 
perature is  the  same  as  with  inversion  by  dissolved  enzyme. 

As  is  well  known,  invertase  attacks  the  trisaccharide,  raffinose, 
decomposing  the  cane-sugar  group  present.  This  hydrolysis 
proceeds  more  slowly  than  that  of  free  cane-sugar;  it  has  been 
investigated  by  H.  E.  Armstrong  and  Glover  (Proc. 
Roy.  Soc.,  1908,  80,  317). 


MALTASE 

As  with  emulsin,  so  also  with  maltase,  E.F.Armstrong 
(Proc.  Roy.  Soc.,  1904,  73,  508)  found  the  reaction-constants  of 
the  first  order  to  diminish  considerably. 


Maltose,  5%. 


Hours. 

X 

k.  10* 

Hours. 

X 

k.W* 

1 

7.3 

329 

1 

4-7 

209 

2 

13-9 

325 

3 

11-7 

180 

4 

24-4 

304 

5 

17-8 

170 

7-25 

31-7 

229 

23 

23-9 

52 

23 

35-2 

82 

28 

25-0 

45 

47 

31-4 

35 

Maltose,  10% 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       167 


In  striking  contrast  to  these  are  the  numbers  of  Henri 
and  Mdlle.  P  h  i  1  o  c  h  e  (Soc.  Biol.,  1904,  57,  171),  of  H  e  n  r  i 
and  of  Terroine  (Archivio  di  Fisologia,  1904,  2,  1),  who 
found  that  the  constants  k  for  a  unimolecular  reaction  at  first 
rise,  whilst  the  constants  kn  for  a  definite  initial  concentration 
remain  comparatively  constant. 


Terroine   (loc.  cit.,  p.  4). 
Maltose,  4%. 

P  h  i  1  o  c  h  e   (loc.  cit.,  p.  6). 
Maltose,  4%. 

Minutes. 

fc.lO*. 

2kH.W5. 

Minutes. 

X 

a, 

k.  105. 

50 

88 

167 

63 

0-176 

134 

112 

86 

156 

120 

0-312 

135 

175 

103 

170 

181 

0-441 

139 

230 

127 

202 

241 

0-588 

163 

349 

119 

176 

363 

0-753 

167 

470 

134 

184 

480 

0-824 

157 

588 

122 

166 

600 

0-859 

142 

780 

113 

148 

750 

0-869 

117 

903 

106 

148 

930 

0-900 

107 

The  constants  vary,  however,  with  the  dilution  of  the  mal- 
tase,  so  that  Henri  first  employed  formula  (3),  p.  129,  for  the 
calculation  (m  =  3,  w=l),  but  as  the  agreement  with  the  exper- 
imental data  was  not  satisfactory,  he  proposed  the  introduction 
of  new  constants  into  this  formula. 

Further,  H  e  r  z  o  g  (Zeitschr.  f.  allg.  Physiol.,  1904,  4, 
177)  obtained  different  results,  which  he  calculated  according 
to  formula  (le),  p.  129,  with  variable  coefficients. 


Maltose,  3-67%.  «=1-12. 


Maltose,  3-217%.     e=2. 


Minutes. 

X 

a 

k 

*i(l-e)10». 

Minutes. 

X 

a 

t 

&i(l-e)io«. 

30 

0-128 

195 

27 

20 

0-066 

149 

16 

60 

0-215 

175 

23 

40 

0-138 

161 

19 

120 

0-328 

144 

22 

80 

0-193 

116 

15 

180 

0-415 

127 

22 

120 

0-248 

103 

14 

240 

0-471 

115 

20 

180 

0-312 

90 

15 

370 

0-567 

76 

21 

240 

0-358 

80 

15 

590 

0-631 

73 

20 

620 

0-687 

81 

22 

168 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


It  is  very  probable  that  the  action  of  maltase,  like  invertase- 
action,  depends  on  the  concentration  of  the  H'-ions  present. 
It  is  most  desirable  that  a  new  investigation  should  be  made  in 
which  this  influence  is  considered;  more  simple  laws  of  reaction 
would  then  probably  be  found  to  hold. 

According  to  Croft  Hill  and  to  Lintner  and  K  r  6  b  e  r 
(Chem.  Ber.,  1895,  28,  1050)  the  optimum  temperature  is  40°.  A  s  p  e  r  - 
g  i  1 1  u  s  -  maltase  is  stated  to  be  only  slightly  sensitive  towards  chloro- 
form (H  6  r  i  s  s  e  y  ,  Soc.  Biol,  1896,  48,  915).  In  working  with  yeast- 
maltase  Fischer  recommends  the  use  of  toluene. 


LACTASE 

Quantitative  measurements  have  been  made  by  E  .  F  .  A  r  m  - 
strong  (Proc.  Roy.  Soc.,  1904,  73,  506). 

In  the  various  series  of  experiments,  the  reaction-constants 
of  the  first  order  diminish  considerably,  but  not  regularly.  As 
examples,  the  following  tables  may  be  given- 

Two  GRMS.  MILK-SUGAR  PER  100  c.c. 


I. 

II. 

100  c.c.  enzyme-extract. 

40  c.c.  enzyme-extract. 

Hours. 

X 

fc.lO< 

Hours. 

X 

£.10* 

1 

22-1 

1085 

0-33 

3-2 

423 

2 

31-2 

812 

0-66 

6-4 

430 

3 

38-9 

713 

1 

9-6 

438 

4 

45-8 

665 

1-5 

13-2 

410 

5 

51-5 

629 

2 

16-4 

389 

6 

56-6 

664 

3 

20-8 

338 

10 

69-0 

509 

5 

25-2 

252 

17 

84-2 

471 

23 

47-6 

122 

23 

92-4 

461 

100 

89-6 

82 

The  constants  are  evidently  dependent  on  the  concentration 
ratio,  enzyme  :  substrate.  With  a  relatively  large  amount  of 
enzyme,  the  constant  diminishes  continuously,  but  if  less  enzyme 
is  present,  equal  amounts  of  sugar  are  at  first  hydrolysed  in 
equal  intervals  of  time. 

The  following  table  shows  the  influence  of  the  concentration 
of  lactase  on  the  velocity  of  hydrolysis  of  a  5%  milk-sugar  solu- 
tion; the  quantities  of  sugar  hydrolysed  are  given  in  percentages: 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       169 


C.c.  Lactase. 

1  -5  hour. 

20  hours. 

25  hours. 

45  hours. 

68  hours. 

1-0 

0-15 

2-2 

2-6 

3-9 

4-8 

2-5 

0-4 

5-8 

6-8 

10-2 

12-6 

10. 

1-6 

23-3 

— 

38-6 

48-5 

20. 

3-2 

45-8               54-5 

— 

— 

1 

The  quantities  hydrolysed  are  approximately  proportional 
to  the  enzyme-concentrations,  so  long  as  these  are  not  too  high. 
As  is  shown  by  the  next  table ,  very  small  amounts  of  enzyme 
are  able  to  hydrolyse  only  small  amounts  of  sugar;  their  activity 
then  ceases,  indicating  that  the  products  of  hydrolysis,  glucose 
and  galactose,  combine  with  the  enzyme  and  so  withdraw  it 
from  the  reaction  with  the  substrate. 

5%  Milk-sugar  solution;  amounts  hydrolysed  in  percentages. 


c.c.  Lactase. 

24  hours. 

144  hours. 

0-66 

2-3 

2-3 

1-0 

3-2 

3-5 

2-0 

6-3 

7-4 

5-0 

15-4 

34-0 

If  the  quantity  of  milk-sugar  is  varied,  it  is  found  that,  with 
large  proportions  of  enzyme,  the  amount  hydrolysed  in  unit 
time  is  proportional  to  the  concentration  of  the  sugar,  so  that  the 
values  of  k  are  equal. 

PERCENTAGES  OF  MILK-SUGAR  HYDROLYSED 


Milk-sugar 
per  100  c.c. 

Hydrolysed 
after  3  hours. 

k.  10*. 

1-0  grm. 

0-5     " 

0-185 
0-098 

296 

298 

0-2     " 

0-0416 

337       . 

For  comparatively  large  amounts  of  substrate  the  percentage 
of  sugar  hydrolysed  is  inversely  proportional  to  its  concentration, 
so  that  in  unit  time  equal  absolute  amounts  of  sugar  are  decom- 
posed, no  matter  what  the  concentration. 


170 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


According  to  H.  E.Armstrong,  E.  F.  Armstrong 
and  H  o  r  t  o  n  ,  emulsin  contains  a  gluco-lactase.  In  a  series  of 
experiments  carried  out  by  Armstrong  (Proc.  Roy.  Soc., 
1904,  73,  507)  with  milk-sugar  and  emulsin,  the  values  of  k 
fell  rapidly. 


2  grms.  lactose 


2  grms.  lactose       \  per  100  c.c. 


0  -2  grm.  emulsin   /*" 

0-4  grm.  emulsin  ^^ 

Minutes. 

X 

&.10*. 

X 

vr 

Minutes. 

X 

k.  10*. 

X 

~VT 

0-5 

3-2 

282 

4.5 









1-0 

4-8 

214 

4-8 

1-0 

4-9 

218 

4-9 

2-0 

6-4 

143 

4-5 

2-0 

7-5 

169 

5-3 

3-0 

7-6 

114 

4-4 

— 

— 

— 

— 

4-5 

9-0 

91 

4-2 

4-5 

9-4 

95 

4-4 

6-0 

10-0 

91 

4-1 

6-0 

10-6 

81 

4-3 

23-0 

19-7 

41 

4-1 

23-0 

30-5 

69 

2-0 

29-0 

22-0 

37 

4-1 

29-0 

35-0 

64 

2-0 

48-0 

29-0 

31 

4-2 

48-0 

47-8 

59 

2-2 

53-0 

30-7 

30 

4-2 

53-0 

50-0 

57 

2-2 

144-0 

62-2 

29 

5-2 

144-0 

84-0 

55 

7-0 

264-0 

77-5 

24 

4-8 

No  simple  relation  between  the  time  and  amount  of  reaction 
is  at  first  evident  from  these  figures,  and  the  tables  given  in 
this  paper  indicate  none  between  the  velocity  of  reaction  and  the 
concentration  of  enzyme.  The  diminution  of  the  constant  may 
be  explained  by  the  retarding  influence  of  the  products  formed 
by  the  reaction. 

Later  investigations  (Proc.  Roy.  Soc.,  1908,  80,  326)  show 
good  agreement  with  a  unimolecular  reaction,  if  relatively  large 
quantities  of  enzyme  (almond-extract)  are  employed. 

5%  Milk-sugar  solution 


Hours. 

40  c.c.  enzyme-solution. 

60  c.c.  enzyme-solution. 

X 

fc.KM. 

X 

&.10*. 

2 

15-2 

358 

18-5 

444 

3 

18-5 

296 

22-7 

373 

5 

21-1 

251 

36-5 

394 

7 

33-5 

253 

44-9 

380 

9 

42-2 

264 

52-5 

360 

10 

44-0 

252 

53-5 

332 

24 

73-4 

240 

76-0 

258 

EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       171 

As  is  seen  from  the  third  and  fifth  columns,  there  is  here 
also  no  proportionality  between  concentration  of  enzyme  and 
velocity.  Only  relatively  small  amounts  of  enzyme  have  hydro- 
lysing  actions  proportional  to  their  concentrations,  as  is  shown 
by  the  following  figures: 

Amount  of  enzyme 10        20        40  60  c.c. 

Velocity  constant,  /c.104 107      212      279         385 

Like  invertase  and  maltase,  lactase  exhibits  its  optimal  activity  in 
faintly  acid  solution:  according  to  B  i  e  r  r  y  and  S  a  1  a  z  a  r  (C.  R., 
1904,  139,  381),  with  0-002-0-004%  of  HC1,  which  corresponds  with  a 
concentration  of  hydrogen-ions  of  about  10~3.  Lactic  acid  is  stated 
by  Bokorny  (Maly's  Jahrb.,  1903,  33)  to  exert  a  specific  accelerating 
influence. 


ENZYMES   OF  EMULSIN 

1.  (i-G  lucosidase 

The  first  quantitative  investigation  of  emulsin  was  made 
byTammann  (H.,  1891,  16,  298  et  seq.),  who  examined 
the  action  of  this  enzyme  on  amygdalin,  salicin,  arbutin  and 
coniferin.  As  the  following  comparison  of  the  calculated  and 
observed  values  of  (a—x)  shows,  at  25°  the  process  appears  to  be 
unimolecular : 

DECOMPOSITION  OF  SALICIN  BY  EMULSIN 


t  (hours). 

a  —  x 

ft 

found. 

calc. 

1 

87 

88 

0-061 

3 

68 

67 

0-057 

5 

42 

52 

0-075 

8 

35 

35 

0-058 

12 

24 

21 

0-052 

26 

9 

3 

0-040 

Important  progress  is  marked  by  the  work  of  Hudson 
and  Paine  (Journ.  Amer.  Chem.  Soc.,  1909,  31,  1242)  on  the 
decomposition  of  salicin.  These  authors  paid  attention  to  the 
facts  that  the  hydrolysis  of  salicin  yields  ^-glucose  and  that  the 
reaction  is  extremely  sensitive  towards  hydrogen  and  hydroxyl- 


172 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Ions.  The  following  numbers  show  that,  under  suitable  exper- 
imental conditions,  satisfactory  constancy  of  the  velocity  constant 
k  of  the  first  order  is  obtained. 


Minutes. 

Specific  rotation 
(alkaline  solution). 

IWk. 

0 

-62-0° 

— 

Temperature,  30° 

10 

OT:  *  O 

360 

20 

-48-7 

330 

Concentration  of 

30 

-41-6 

353 

salicin, 

35 

-39-5 

339 

5% 

85 

-15-8 

374 

145 

+  2-9 

350 

00 

+32-2 

~~~ 

The  influence  of  acids  and  bases  is  indicated  by  the  following 
table: 


Concentration  of 
NaOH. 

Activity  of  the 
emulsin. 

1 

Concentration  of 
HC1. 

Activity  of  the 
emulsin. 

0-005 

0 

0-00027 

222 

0-0009 

138 

0-0005 

225 

0-0005 

195 

0-0018 

242 

0-00009 

222 

0-005 

255 

0-009 

206 

The  optimal  activity  is  hence  shown  with  0-005  grm.-mols. 
of  HC1  per  litre. 

A  u  1  d '  s  experiments  (Journ.  Chem.  Soc.,  1908,  93,  1251) 
on  the  hydrolysis  of  salicin  by  an  enzyme  (phaseolunatase) 
present  in  Phaseolus  lunatus  also  seem  to  indicate 
constancy  of  the  values  of  k  (Table  III). 


Hours. 

X 

k 

Hydrolysis  of  salicin  by  phaseolunatase 
at  39-5°  

0-5 
1  0 

15-8 
30-9 

141 
145 

2-0 
3-0 

59-0 
6*7 

156 
147 

H  e  r  z  o  g  (K.  Akad.  v.  Wetensch.,  Amsterdam,  Sitzungsber., 
1903,  and  Zeitschr.  f.  allg.  PhysioL,  1904,  4,  163)  has  likewise 
made  experiments  on  the  decomposition  of  salicin  by  emulsin: 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       173 


Minutes. 

X 

a 

fc.105. 

10<  .       o  -  (X 
kH=~lOS^C 

Temp.  25°. 

24 

0-174 

346 

15 

54 

0-354 

351 

16 

Salicin  solution, 

86 

0-450 

302 

'     14 

0-07  N. 

210 

0-691 

243 

13 

270 

0-775 

239 

14 

s=0-6 

371 

0-847      . 

219 

14 

2.  Amygdalase   and    Hydroxynitrilase 

The  investigations  of  Armstrong,  on  the  one  hand,  and 
Rosenthaler,  on  the  other,  indicate  that  the  name 
amygdalase  should  be  given  to  that  enzyme  which  hydrolyses 
amygdalin  into  mandelonitrile  glucoside  and  glucose.  A  g- 
glucosidase  present  in  "  emulsin  "  then  decomposes  the  mandelo- 
nitrile glucoside  further  into  glucose  and  mandelonitrile,  and  the 
latter  product  is  finally  broken  down  into  benzaldehyde  and 
hydrocyanic  acid  by  the  hydroxynitrilase.  So  that  three  enzymes 
take  part  in  the  hydrolysis  of  amygdalin.  It  can,  therefore, 
hardly  be  expected  that  the  formation  of  the  final  products 
should  correspond  with  a  simple  reaction-formula. 

The  first  investigation  of  the  system  amygdalin-emulsin 
was  made  by  T  a  m  m  a  n  n  ;  certain  of  his  experiments  on  the 
retardation  of  the  reaction  by  the  products  formed  have  already 
been  referred  to  in  the  preceding  section  (p.  139). 

Auld  (Journ.  Chem.  Soc.,  1908,  93,  1251)  has  recently 
made  a  very  thorough  investigation  of  the  hydrolysis  of  amygdalin. 
He  followed  the  reaction  by  titrating  the  liberated  hydrocyanic 
acid  with  iodine  and  found  increasing  values  for  the  unimolec- 
ular  constant: 

300  c.c.  2%  amygdalin  solution+15  c.c.  2%  emulsin  solution. 


Minutes. 

X 

fc.105. 

10 

6-1 

255 

80 

45-2 

295 

Temperature  40° 

100 

54-9 

309 

150 

75-3 

347 

1360 

93-3 

(x  indicates  the  quantities  of  amygdalin  decomposed,  in  percentages.) 


174 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


H  .  E  .  and  E.  F.  Armstrong  and  H  o  r  t  o  n  (Proc. 
Roy.  Soc.,  1908,  80,  330)  give  their  results  in  the  form  of  the 
following  curves  (Fig.  4) : 

The  curve  representing  the  glucose  formed  is  not  coincident 
with  that  showing  the  hydrocyanic  acid  liberated,  which  would 
be  understandable  if  these  two  substances  were  set  free  in  two 
different  reactions  effected  by  two  different  enzymes.  These 
authors  have  therefore  done  right  in  not  calculating  the  reaction- 


HYDROLYSIS  OF  AMYGDALIN 
BY  EMU  LSI  NAT  25° 


10         12         14 
Time  in  Hours 

FiG.  4. 


constants  for  the  complex  of  reactions   comprised  in  the  hydro- 
lysis of  amygdalin. 

Similar  results  are  given  by  A  u  1  d  (loc.  cit.,  p.  1268)  for  the 
temperature  41°. 

Quantity  of  enzyme 2        3          4          6         12        25        50  c.c. 

Percent,  hydrolysis  in  10 mins.    3-7     6-05     8-7     12-5     17-2    21-6     24-1 

If  dilute  amygdalin  solutions  are  employed,  the  constants 
are,  as  they  should  be  theoretically,  independent  of  the  concen- 
tration of  amygdalin  (A  u  1  d  ,  loc.  cit.,  p.  1270). 

Hydrocyanic  acid  and  glucose  produce  retarding  effects. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       175 

Mention  must  finally  be  made  of  Henri  and  L  a  1  o  u  '  s 
polarimetric  experiments  (Soc.  Biol.,  1903,  55,  868)  on  the 
simultaneous  decomposition  of  amygdalin  and  salicin. 


t 
minutes. 

2% 
salicin. 

2-5%. 
amygdalin. 

2%  salicin 
+2-5% 
amygdalin 

4% 
salicin. 

1-25% 
amygdalin. 

46 

0-67 

0-97 

1-05 

1-08 

0-90 

130 

1-58 

2-38 

3-63                2-25 

1-57 

268 

2-32 

3-15 

4-22 

3-45 

1-56 

GO 

3-15 

3-17 

6-32                6-30 

1-59 

It  will  be  seen  that  the  hydrolysis  of  the  mixture  takeb  place 
much  more  slowly  than  that  of  the  two  constituents  separately. 
This  fact  also  indicates  combination  of  enzyme  and  substrate. 

The  enzyme  is  moderately  resistant  to  chloroform  and  toluene  and 
its  optimum  temperature  is  given  as  45°. 


PROTEOLYTIC   ENZYMES 

The  first  accurate  experiments  on  the  time-course  of  the 
decomposition  of  protein  by  pepsin  are  due  toE.  Schiitz 
(H.,  1885,  9,  577).  They  were  carried  out  with  solutions  of 
globulin-free  egg-albumin  (about  1  grm.  per  10  c.c.),  to  which 
were  added  5  c.c.  of  5%  hydrochloric  acid  and  a  pepsin  solution 
of  definite  strength;  the  solutions  were  then  diluted  to  100  c.c. 
and  kept  at  37-5°  for  16  hours.  The  albumin  was  then  removed 
from  the  solutions  and  the  amounts  of  peptone  formed  determined 
by  means  of  the  optical  rotations.  In  this  way  S  c  h  ii  t  z  found 
the  velocity  of  digestion  to  be  proportional  to  the  square-root 
of  the  concentration  of  the  pepsin.  The  results  of  the  first  of 
the  three  series  of  experiments  are  given  here : 


Quantity  of 

Rotation  of  the  peptone  in  minutes. 

pepsin. 

Observed  (mean). 

Calculated. 

1 

7-3 

7-4 

2 

9-75 

10-4 

3 

12-8 

12-7 

4 

14-8 

14-7 

5 

16-5 

16-4 

6 

18-45 

18-9 

176 


.    GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Mention  must  also  be  made  of  the  experiments  ofBorissow 
with  trypsin  and  of  Samojloff  with  pepsin,  these  exper- 
imenters also  arriving  at  the  relation  x  =  K^/Et.  (Dissertation, 
St.  Petersburg,  1901;  Arch,  des  Sci.  BioL,  1893,  2,  699;  see 
P  a  w  1  o  w  ,  Arbeit  der  Verdauungsdriisen) ;  there  was  here  no 
intention  of  obtaining  a  representation  of  the  chemical  dynamics 
of  enzyme  action  and,  owing  to  the  experimental  methods  employed 
in  these  investigations,  no  conclusions  concerning  this  can  be 
drawn.  The  same  may  be  said  of  Walther's  researches 
(Arch,  des  Sci.  BioL,  1899,  7,  15). 

Very  extensive  numerical  data  on  the  digestion  of  protein 
by  pepsin  were  given  in  1895  by  J.  S  j  6  q  v  i  s  t  (Skand.  Arch, 
f.  PhysioL,  1895,  5,  317),  who  followed  the  course  of  the  digestion 
by  measuring  the  electrical  conductivity.  Every  100  c.c.  of 
solution,  0-05N  with  reference  to  hydrochloric  acid,  contained 
2-23  grins,  of  albumin  (almost  freed  from  salts  by  dialysis) 
and  also  2-5,  5,  10  or  20  c.c.  of  pepsin  solution.  The  con- 
ductivity of  these  solutions  fell  during  the  experiment  from  the 
initial  value  [i  =  188-4  (old  units)  to  a  final  value  of  about  83-4. 
The  amount  of  albumin  acted  on,  x,  was  taken  as  proportional 
to  the  fall  A  in  the  conductivity.  The  following  tables  contain 
the  observed  values  of  A  at  37°,  together  with  the  corresponding 

2-5  c.c.  of  pepsin  solution  per  100  c.c. 


Hours. 

Conductivity, 

M 

Change  of 
conductivity, 
A 

X 

*4 

k  -  — 

H'S  —        /  — 

Vt 

0 

188-4 

_ 







0-5 

— 

— 

— 

— 

— 

1 

— 

— 

—   . 

— 

— 

2 

177-3 

11-1 

10-5 

2-97 

7-45 

4 

171-1 

17-3 

16-41 

3-78 

8-21 

6 

167-4 

21-0 

19-93 

3-81 

8-13 

8 

164-5 

(23-9) 

22-68 

3-77 

8-02 

9 

163-1 

25-3 

24-00 

3-82 

7-90 

12 

159-9 

28-5 

27-04 

3-70 

7-70 

16 

156-4 

(32-0) 

30-36 

3-62 

7-59 

20 

152-9 

35-5 

33-68 

3-70 

7-53 

32 

146-2 

42-2 

40-04 

3-40 

7-08 

48 

139-8 

(48-6) 

45-06 

3-20 

6-50 

64 

135-0 

(53-4) 

50-78 

3-13 

6-34 

96 

127-9 

60-5 

57-41 

2-80 

5-87 

00 

3-49 

EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS   177 


proportional  values  of  x,  the  calculated  values  of  the  constant 
KA  of  the  A  r  r  h  e  n  i  u  s  formula  (18)  or  (20)  and,  finally,  the 
values  of  the  constant  of  S  c  h  ii  t  z  's  formula  x  =  Ks-\/t. 

5  c.c.  of  pepsin  solution  per  100  c.c. 


Hours. 

Conductivity, 

M 

Change  of 
conductivity, 
A 

X 

KA 

X 

KS  =  —  -7=- 

Vt 

0 

188.4 





_ 

_ 

0-5 

— 

— 

— 

— 

— 

1 

178-2 

10-2 

9-68 

4-93 

9-8 

2 

172-8 

15-6 

14-80 

6-09 

10-5 

4 

164-7 

23-7 

22-49 

7-49 

11-2 

6 

159-5 

28-9 

27-42 

7-70 

11-2 

8 

155-5 

(32-9) 

31-22 

7-62 

11-0 

9 

153-5 

(34-9) 

33-11 

7-88 

11-0 

12 

149-4 

39-0 

36-58 

7-47 

10-6 

16 

145-2 

(43-2) 

40-15 

8-25 

10-0 

20 

141-0 

47-4 

44-98 

7-39 

10-0 

32 

133-1 

(55-3) 

52-47 

6-85 

9-3 

48 

126-2 

(62-2) 

59-02 

6-30 

8-5 

64 

121-4 

(67-0) 

63-58 

5-85 

7-9 

96 

114-4 

74-0 

70-21 

5-21 

7-3 

00 

— 

— 

— 

6-84 

10  c.c.  of  pepsin  solution  per  100  c.c. 


Hours. 

Conductivity, 

M 

Change  of 
conductivity, 
A 

X 

KA 

X 

KS=      ,— 

Vt 

0 

188-4 









0-5 

179-2 

9-2 

8-73 

8-04 

12-35 

1 

174-2 

14-2 

13-47 

10-34 

13-47 

2 

165-9 

22-5 

21-35 

13-40 

15-10 

4 

154-8 

33-6 

31-88 

16-30 

15-94 

6 

148-0 

40-4 

38-34 

16-65 

16-01 

8 

143-2 

(45-2) 

42-88 

16-42 

15-31 

9 

140-8 

47-6 

45-14 

16-51 

15-05 

12 

136-1 

52-3 

49-62 

14-09 

14-32 

16 

130-9 

(57-5) 

54-56 

15-23 

13-64 

20 

125-7 

62-7 

59-50 

15-44 

13-30 

32 

119-4 

69-0 

65-46 

12-90 

11-60 

48 

113-1 

75-3 

71-45 

11-25 

10-31 

64 

109-1 

(79-3) 

75-25 

10-08 

9-41 

96 

101-8 

86-6 

82-17 

9-60 

8-39 

00 

— 

— 

— 

13-30 

178 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


20  c.c.  of  pepsin  solution  per  100  c.c. 


Hours. 

Conductivity, 

M 

Change  of 
conductivity, 
A 

X 

KA 

X 
KS  =  —  7= 

Vt 

0 

188-4 



— 



— 

'0.5 

176-0 

12-4 

11-77 

15-0 

16-64 

1 

167-8 

20-6 

19-55 

22-2 

19-55 

2 

157-9 

30-3 

28-75 

25-9 

20-33 

4 

144-5 

43-9 

41-66 

30-6 

20-83 

•    6 

137-2 

51-2 

48-58 

29-7 

19-83 

8 

133-0 

(55-4) 

52-57 

25-1 

18-54 

9 

130-1 

58-3 

55-52 

28-0 

18-44 

12 

125-8 

62-6 

59-40 

25-6 

17-15 

16 

121-6 

(66-8) 

63-34 

25-1 

15-81 

20 

117-3 

71-7 

68-03 

22-5 

15-21 

32 

109-8 

78-6 

74-58 

19-5 

13-18 

48 

102-3 

86-1 

81-70 

18-3 

11-79 

64 

97-4 

(91-0) 

86-34 

14-4 

10-49 

96 

91-2 

97-2 

92-24 

18-0 

9-41 

oo 

— 

— 

— 

22-7 

S  j  6  q  v  i  s  t  's  observations  hence  indicate  that  the  relation 
x  =  Ks\/t  holds  moderately  well  during  the  first  half  of  the  reaction. 

Calculation  of  the  constants  for  the  various  concentrations 
of  enzyme  E  shows  that  these  are  approximately  in  the  propor- 
tions, A/0^2  :  VoT :  VtTo^ :  Vo-025. 

KS.^ 7-6  10-4          14-4          18-2 

VE 0-500          0-707        1-00          1-42 

Quotient  KS  :  VE    15-2  14-7          14-4          12-8 

So  that,  for  small  and  equal  values  of  t,  S  c  h  ii  t  z  's  rule 
holds,  i.e.,  the  quantity  of  substance  transformed  is  inversely 
proportional  to  the  square-root  of  the  concentration  of  the  enzyme. 
The  proportionality  between  the  amount  of  albumin  hydrolysed 
and  the  square-root  of  Et  (enzyme-concentration  X  time)  is 
much  more  general,  as  is  shown  by  the  following  table  in  which 
Arrhenius  (Immunochemistry,  p.  67)  has  collected  to- 
gether the  diminutions  of  conductivity  given  by  S  j  6  q  v  i  s  t. 

Et=              0-05    0-1  0-2  0-4  0-8  1-6  3-2      4-8      6-4  9-6 

#  =  0-025  11-1     17-3  23-9  32-0  42-2  53-4  —        —        —  — 

0-05    10-2    15-6  23-7  32-9  43-2  55-3  67-0  74-0      —  — 

0-01       9-2     14-2  22-5  33-6  45-2  57-5  69-0  75-3  79-3  86-6 

0-02      —      12-4  20-6  30-3  43-7  55-4  66-8  73-6  78-6  86-1 


Mean.  .  .  .  10-2 
Calculated  11 


14-9 
15-6 


22-7 
22 


32-2 
31-1 


43-8 
44 


55-4    67-6    74-3     79-0 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       179 

Further  investigation  of  peptic  digestion  is  due  to  Julius 
Schutz  (H.,  1900,  30,  1),  who  coagulated  the  undigested 
protein  remaining  after  15  hours  and  determined  the  nitrogen  in 
the  filtered  liquids  by  K  j  e  1  d  a  h  1  '  s  method.  At  a  tem- 
perature of  38°,  he  obtained  the  following  quantities  of  hydrolysed 
protein  (£0bs.)»  the  values  calculated  from  Schiitz's  rule 
being  given  in  the  final  column. 


Quantity  of  pepsin. 

104.Zobs. 

104.Zcaic. 

1 

212 

213 

4 

471 

426 

9 

652 

639 

16 

799 

852 

25 

935 

1065 

36 

1031 

1278 

In  the  same  year  E  .  Schutz  and  H  u  p  p  e  r  t  (Pfliig. 
Arch.,  1900,  80,  470)  gave  further  data  concerning  peptic  diges- 
tion. The  decomposition  products — termed  secondary  albumoses 
— of  the  protein  were  determined  polarimetrically.  "  The  quan- 
tities of  secondary  albumoses  formed  are  proportional  to  the 
square-roots  of  the  times."  Further,  the  quantities  of  digested 
protein,  the  sum-totals  of  the  intermediate  products  and  the 
amounts  of  secondary  albumoses,  are  in  the  same  ratios  as  the 
amounts  of  protein  employed,  namely,  1:2:3:4. 

E  .  Schutz  and  H  u  p  p  e  r  t  also  investigated  the  influence 
of  hydrochloric  acid.  Under  certain  conditions,  secondary 
albumoses  are  formed  in  proportion  to  the  quantity  of  protein, 
to  the  square-root  of  the  time,  and  to  the  concentrations  of  pepsin 
and  acid.  The  conditions  for  this  rule  to  hold  are  a  moderately 
rapid  reaction  and  a  concentration  of  acid  not  exceeding  0  •  2%. 

If  we  denote  the  amount  of  secondary  albumoses  by  S,  that 
of  albumin  by  A,  the  time  by  t,  the  concentration  of  hydrochloric 
acid  by  s,  and  the  quantity  of  pepsin  by  P,  the  velocity  with 
which  the  secondary  albumoses  are  formed  is  expressed  by 


It  must  be  stated  that  objections  have  been  raised  by 
Sjoqvist  (loc.  cit.)  to  the  methods  used  in  these  experi- 
ments. 


180 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Gross  (Berl.  klin.  Wochens.,  1908,  45,  643)  expressed 
the  view  that  S  c  h  (i  t  z  's  rule  does  not  hold  for  peptic  diges- 
tion, but  that  the  amount  of  digestion  is  proportional  directly 
to  the  quantity  of  enzyme  and  inversely  to  the  time  of  digestion. 
His  experiments  were  carried  out  as  follows:  Increasing  amounts 
of  pepsin  were  added  to  constant  quantities  of  an  acid  solution 
of  casein,  observation  being  then  made  in  each  case  of  the  time 
when  the  whole  of  the  casein  was  digested,  i.e.,  when  no  turbidity 
was  produced  by  addition  of  saturated  sodium  acetate  solution. 
The  observations  were  made  at  intervals  of  10-20  seconds,  the 
temperature  being  40°. 

The  following  series  of  results  may  be  quoted : 


Casein  solution,  50  c.c. 

Casein  solution,  50  c.c. 

Gastric  juice, 
c.c. 

Digestion  complete 
in  minutes. 

Griibler  pepsin, 
(0-1%)  c.c. 

Digestion  complete 
in  minutes. 

1-0 

52-7 

1-0 

64-0 

2-0 

25-0 

2-0 

31-7 

4-0 

12-2 

4-0 

16-7 

8-0 

6-25 

— 

— 

In  order  to  test  Gross's  results,  Kurt  Meyer  (Berl. 
klin.  Wochens.,  1908,  45,  1485)  made  a  number  of  experiments 
by  F  u  1  d  '  s  edestin  method.1  The  quantity  of  pepsin  in  any 
tube  was  four  times,  and  that  of  protein  twice,  that  in  the  pre- 
ceding tube.  M  e  y  e  r  collects  his  results  in  two  tables,  of 
which  one  is  given  here: 


1%  edestin  solu- 

1% pepsin  solution  (G  r  u  b  1  e  r)  in  0-03  HC1. 

tion  in  0-03  HC1. 

c.c. 

0-0025  c.c. 

0-01  c.c. 

0-04  c.c. 

0-16  c.c. 

0-1 

4. 





_ 

0-2 

+ 

— 

— 

0-4 

-f. 

_ 

0-8 

-f 

1-6 

3-2 

1  The  method  was  so  modified  that  the  amounts  of  digestion  could  be 
obtained  in  one  series  of  experiments;  thus,  series  with  increasing  quantities 
of  edestin  and  similar  series  with  increasing  quantities  of  pepsin  were  carried 
out. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS        181 


Incipient  turbidity  is  indicated  by  +. 

No  exact  idea  can  be  formed  of  the  magnitude  of  the  experr 
imental  error  is  these  investigations.  K  .  Meyer  himself, 
however,  draws  the  conclusion  that  S  c  h  ii  t  z  '  s  rule  is  valid 
for  peptic  digestion. 

0-05  c.c.  gastric  juice. 


Quantity  of  casein, 
c.c. 

Time  of  digestion 
in  minutes. 

Quantity  of  casein, 
c.c. 

Time  of  digestion 
in  minutes. 

5 

6-3 

10 

13-3 

6 

7-5 

12 

14-3 

7 

8-7 

14 

17-0 

8 

10-0 

16 

21-2 

For  the  sake  of  completeness  it  may  be  mentioned  that  S  p  r  i  g  g  s 
(Journ.  of  PhysioL,  1902,  35,  465)  followed  the  course  of  pepsin-action 
by  measurements  of  the  viscosity  with  0  s  t  w  a  1  d  '  s  viscosimeter. 
Against  these  experiments  the  objection  may,  however,  be  raised  that 
the  relation  of  viscosity  to  the  degree  of  protein  hydrolysis  is  not  suffi- 
ciently known,  so  that  no  safe  conclusions  can  be  drawn  from  the  results 
of  these  measurements. 

W  e  i  s  (Medd.  fra  Carlsberg  Lab.,  1903,  5,  127)  has  made  a 
very  thorough  investigation  of  the  action  of  vegetable  proteases. 
He  found  that  peptic  action  proceeds  relatively  rapidly,  whilst 
the  tryptic  decomposition  of  the  albumoses  is  more  gradual; 
the  two  actions  can,  to  some  extent,  be  separated.  From  the 
results  obtained,  which  are  extremely  difficult  to  deal  with  in 
detail,  it  is  to  be  concluded  that,  for  the  proteolysis  of  vegetable 
protein — at  any  rate  within  a  certain  region  of  concentration — 
S  c  h  u  t  z  '  s  rule  appears  to  hold.  According  to  the  results 
given  on  p.  176,  etc.,  the  exponent  of  the  enzyme -concentration 
increases,  with  increasing  dilution  of  the  enzyme,  from  0-5 
(Schiitz  's  rule)  to  1. 

In  the  separate  series  of  experiments,  the  amounts  of  sub- 
stance transformed  are  proportional  to  the  square-roots  of  the 
times.  From  the  table  on  p.  183  of  the  above  paper  we  extract 
the  following  numbers,  the  values  of  x:\/T  being  given  in  addi- 
tion. 


182 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Amount  of  change. 

Amount  of  change. 

Hours. 

Mgrms.  N. 

Ratio^_ 

Hours. 

Mgrms.  N. 

Ratio. 

/  

t 

X 

x  :  -\t 

t 

X 

x  :  Vt 

1 

5-34 

5-34 

I 

5-80 

5-80 

2 

8-42 

5-96 

2 

8-54 

6-04 

3 

9-82 

5-67 

3 

(12-00) 

5-50 

4 

11-92 

5-96 

4 

11-34 

5-67 

5 

12-98 

5-81 

5 

12-94 

5-79 

6 

13-70 

5-59 

6 

13-32 

5-44 

9 

17-22 

5-74 

9 

14-20 

4-73 

The  author  has  also  calculated  the  results  of  the  experiments 
in  which  W  e  i  s  varied  the  concentration  a  of  the  substrate  (pro- 
tein). The  third  column  gives  the  constants  k  for  unimolecular 
reactions;  the  fourth,  the  product  ka  and  the  fifth,  the  constant 
KS  of  the  formula  x^/a  =  Ks. 


AFTER  5  HOURS  l 


Protein 

Amount  of  N  trans- 

concentra- 
tion. 

formed  as  percentage 
of  total  N. 

7                1        1                     ° 

k=—  -log  

t           a—x 

k.a.lQG 

Ks 

a 

X 

1% 

36-2 

0-00065 

65 

36-2 

2 

25-9 

0-00043 

86 

36-6 

3 

20-3 

0-00033 

100 

35-2 

4 

16-0 

0-00025 

100 

32-0 

5 

13-2 

0-00020 

102 

29-5 

AFTER  2  HOURS 


Protein 

Amount  of  N  trans- 

concentra- 
tion. 

formed  as  percentage 
of  total  N. 

,        1    ,          a 
k=  log  —  — 

t           a—x 

fc.a.lO" 

Ks 

a 

X 

1% 

22-0 

0-00090 

90 

22-0 

2 

17-0 

0-00067 

134 

24-0 

3 

13-1 

0-00051 

153 

22-7 

4 

9-1 

0-00035 

140 

18-2 

5 

7-9 

0-00030 

150 

17-7 

1  The  numbers  in  the  two  tables  were  obtained  in  two  separate  series  of  experiments 
«md  are  hence  not  comparable. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       183 

It  will  be  seen  that  the  amount  of  protein  changed  diminishes 
with  increasing  values  of  a.  Arrhenius  (Immunochemistry, 
p.  85)  explains  this  as  follows:  When,  for  example,  10%  of  the 
protein  is  digested,  the  absolute  amount  of  the  products  of  the 
reaction  is  doubled  if  the  initial  concentration  a  is  doubled.  But 
the  velocity  of  reaction  is  inversely  proportional  to  the  absolute 
quantity  of  the  products  and,  therefore,  also  to  the  initial  con- 
centration. According  to  what  was  stated  on  p.  133,  the  amount 
of  change,  expressed  as  a  percentage  of  the  total  amount  of  pro- 
tein, is  hence  approximately  inversely  proportional  to  the  square- 
root  of  a,  as  is  shown  by  the  fifth  columns  of  the  two  tables  given 
above. 

The  work  of  W  e  i  s  on  vegetable  proteinases  deals  also  with 
the  trypsins,  that  is,  with  those  proteolytic  enzymes  which  act 
in  alkaline  solution. 

From  the  results  of  experiments  carried  out  virtually  by 
M  e  1 1 '  s  method,  H.M.Vernon  (Journ.  of  Physiol.,  1901, 
26,  421)  has  drawn  the  conclusion  that  the  digestion  of  fibrin 
by  trypsin  follows  S  c  h  ii  t  z  '  s  rule  if  the  times  of  digestion 
are  corrected  for  the  destruction  of  the  trypsin  in  the  soda 
solution. 


Amount  of  enzyme- 
extract  E  in  c.c. 

Time  of  digestion 
t  in  minutes. 

Corrected  time  of 
digestion  in  minutes. 

txVE. 

2 

11-8 

11-14 

16-2 

1 

17-7 

16-25 

16-3 

0-5 

26-8 

23-62 

16-7 

0-25 

36-1 

30-57 

15-3 

0-125 

80-4 

57-32 

20-3 

0-0625 

176-0 

93-57 

23-4 

As  has  long  been  known  qualitatively,  the  concentration  of 
the  acid  present  in  peptic  digestions  exerts  a  marked  influence 
on  the  time-course  of  the  reaction.  This  influence  is  expressed 
quantitatively  in  Schiitz's  formula  mentioned  on  p.  179, 
according  to  which — with  certain  definite  conditions  of  the  enzyme- 
and  substrate-concentrations — the  velocity  of  reaction  is  pro- 
portional to  the  square-root  of  the  concentration  of  the  hydro- 
chloric acid. 


184  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

The  optimal  concentration  of  the  hydrogen-ions  has  been 
the  subject  of  a  recent  and  thorough  investigation  bySorensen 
(Biochem.  Z.,  1909,  21,  288).  In  this  work  the  concentration 
of  the  H-ions  was  determined  electrometrically,  the  progress  of 
the  reaction  being  measured  by  the  amounts  of  protein  pre- 
ciptable  after  different  times  by  stannous  chloride  or  tannic  acid. 
The  results  are  given  in  the  following  table,  the  "  exponents  of 
the  hydrogen-ions,"  PH,  being  given  in  the  first  column;  by  this 
Sorensen  understands  the  logarithm  to  base  10  of  the  recip- 
rocal of  the  normality-factor  of  the  solution  as  regards  hydrogen- 
ions.  The  concentration  of  these  ions  is  given  also  in  the  ord- 
inary form  (column  2). 

It  will  be  seen  that  the  acidity-optimum  increases  with  the 
duration  of  the  peptic  action. 

The  influence  of  the  hydrochloric  acid  must  be  explained,  as 
already  mentioned,  by  the  protein  hydrochloride  being  more 
readily  acted  on  than  the  free  protein.  Further  the  enzyme 
itself  may  be  in  the  form  of  a  salt,  this  pepsin  hydrochloride 
showing  increased  activity;  this  possibility  has  recently  been 
emphasised  by  J.  L  o  e  b  (Biochem.  Z.,  1909,  19,  534)  but  we 
have  no  definite  indications  on  this  question.  As  the  author  has 
pointed  out  (Ergeb.  der  Physiol.,  1907,  6),  the  "  pepsin-hydro- 
chloric acid  " — the  existence  of  which  has  been  so  often  assumed- 
can  mean  nothing  but  a  pepsin  salt  of  hydrochloric  acid. 

The  older  investigations  ofBorissow  on  tryptic  digestion 
have  already  been  mentioned. 

L  .  P  o  1 1  a  k  (Hofm.  Beitr.,  1904,  6,  95)  also  employed 
M  e  1 1 '  s  method  to  examine  a  tryptic  enzyme,  g  1  u  t  i  n  a  s  e  , 
which  he  isolated,  and  which  acts  on  gelatine.  He  arrived  at  the 
result  that  the  action  of  this  enzyme  does  not  correspond  exactly 
with  S  c  h  ii  t  z  '  s  rule,  which,  however,  it  approaches  far  more 
closely  than  does  the  mixture  of  enzymes  of  an  ordinary  pancreas 
infusion  or  Griibler's  trypsin. 

V.  Henri  and  Larguier  des  Bancels  have  inves- 
tigated the  action  of  trypsin  on  gelatine  (C.  R.,  1902,  136,  1581). 
By  regarding,  as  S  j  6  q  v  i  s  t  did,  the  change  of  conductivity 
as  proportional  to  the  progress  of  the  reaction,  they  obtained 
confirmation  of  the  formula 


a-x 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       185 


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186  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Since,  however,  the  final  value  of  the  process  is  not  observed 
(or,  at  any  rate,  not  given)  and  the  observations  are  only  spread 
over  the  very  short  period  of  about  an  hour,  the  real  decomposi- 
tion of  protein  cannot  have  proceeded  very  far  and  these  exper- 
iments tell  little  about  the  course  of  tryptic  digestion.  As  has 
been  calculated  by  Arrhenius,  the  conductivity  is  approx- 
imately proportional  to  the  square-root  of  the  time  of  digestion. 

/  (minutes) 10  20  30  40  50 

Conductivity  (mean) 27-3        44-0        53-0        58-7        65-7 

7-37\A 29-3        41-5        50-0        58-7        68-8 

Of  wider  scope  are  the  experiments  of  B  a  y  1  i  s  s  (Arch. 
Sci.  Biol.  St.  Petersburg,  1904,  11,  Supplement),  in  which  also  the 
conductivity  method  was  employed.  The  substrate  used  was 
partly  caseinate  in  faintly  ammoniacal  solution  and  partly  gelatine. 
The  experimental  results  are  given  mostly  in  the  graphic  form. 
It  is  found  that  the  constants  for  unimolecular  reactions  diminish 
considerably  with  lapse  of  time,  this  being  attributed  to  the  retard- 
ing effect  of  the  products  of  the  reaction.  The  numbers  obtained 
by  B  a  y  1  i  s  s  are  indeed  in  good  agreement  with  the  formula, 

aln- x  =  KEt. 

a  —  x 

With  concentrations  of  casein  up  to  about  4%,  the  velocity  of 
digestion  is  proportional  to  the  concentration  of  the  substrate;  in 
4-8%  solutions,  the  velocity  is  independent  of  the  concentration 
of  the  substrate,  whilst  with  more  than  8%,  inverse  proportionality 
sets  in.  As  regards  the  relation  between  the  velocity  and  the 
concentration  of  the  enzyme,  approximate  proportionality  exists 
during  the  first  quarter  of  the  reaction;  but  even  in  the  second 
quarter,  the  velocity  of  reaction  is  considerably  less  than  would 
correspond  with  the  concentration  of  the  trypsin  (loc.  cit.,  p.  26). 

Using  V  o  1  h  a  r  d  '  s  method  for  estimating  pepsin  and 
trypsin — which  will  be  referred  to  in  the  Appendix —  W.  L  6  h  - 
lein  (Hofm.  Beitr.,  1905,  7,  120)  arrived  at  the  result  that 
tryptic  digestion  does  not  follow  S  c  h  u  t  z  '  s  rule.  0  .  F  a  u  - 
b  e  1'  s  results  (Hofm.  Beitr.,  1907,  10,  35)  point  to  the  same 
conclusion. 

The  measurements  which  have  as  yet  been  made  do  not 
indicate,  with  the  certainty  that  might  be  desired,  the  con- 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       187 

ditions  for  simple  proportionality  between  velocity  of  reaction 
and  the  concentration  of  the  enzyme.  But  far  more  often  than  in 
experiments  with  pepsin  have  the  conditions  been  such  that  the 
relations  : 


Amount  of  digested  protein  =  const. 
and 

Amount  of  digested  protein  =  const.  \/T, 

have  been  found  to  hold  only  over  a  very  limited  range. 

But,  as  with  pepsin  (cf.  p.  179),  it  appears  to  be  quite  general 
that  the  same  quantity  a  of  protein  is  digested  if  the  amonut 
of  enzyme  is  made  to  vary  inversely  with  the  time,  i.e. 

x  =  const.  f(E.t)  .......     (22) 

This  rule  evidently  holds  only  for  those  cases  in  which  the 
proportionality  between  x  and  Et  is  direct.  This  occurs  with 
undisturbed  unimolecular  catalytic  reactions,  so  long  as  the 
products  of  the  reaction  x  are  small  in  amount  compared  with 
the  substrate  a.  Further,  according  to  the  measurements  of 
Bayliss,  Hedin  and  others,  this  rule  is  obeyed,  in  the  case 
of  trypsin,  with  small  quantities  of  enzyme  and  also  with 
conditions  so  chosen  that 

x  =  const.  \/Et. 

This  rule  is  contained  in  the  widely-used  formula  (18),  as  is 
shown  by  the  derivation  of  the  latter. 

Formula  (22)  evidently  means  that  the  progress  of  the  diges- 
tion depends  only  on  the  quantity  of  protein 
already  hydrolysed,  no  matter  whether  this  has  been 
obtained  by  the  action  of  much  enzyme  for  a  short  time  or  by 
that  of  less  enzyme  for  a  longer  time. 

This  relation  has  been  confirmed  by  H  e  d  i  n  (Journ.  of  Physiol., 
1905,  32,  468;  ibid,  1906,  34,  370;  H.,  1908,  57,  471).  One  of  the 
tables  from  the  last  of  these  papers  is  given  below. 

The  amount  of  casein  digested  was  measured  by  the  quantity  of 
nitrogen  not  precipitated  by  tannic  acid.  The  values  given  in  the 
various  columns  under  E.t  indicate  the  numbers  of  tenths  of  a  c.c.  of 
normal  acid  required  to  neutralise  the  ammonia  obtained  by  K  j  e  1  - 


188  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

d  a  h  1 '  s   method  from  equal  volumes  of  the  filtrate  from  the  tannin 
precipitate : 


Et... 
1 

..  1 
5 

•0 

•8 

2-0 
10-35 

2 
12 

•5 
•95 

5-0 
20-15 

7-5 
24-50 

10 

[26 

•0 

•95 

15-0 
31-0 

20-0 
34-05 

2..  .  . 
3.... 
4 

.  .  5 
..  5 
6 

•85 
•70 
•10 

10-75 
10  85 
10-75 

13 
13 
13 

-25 
•35 
•15 

20-40 
20-30 
19-90 

24-65 
24-35 
23-80 

L  *^ 

27 
27 
26 

•8 
•0 
•95 

31-7 
31-05 
30-75 

33-75 
33-55 

As  regards  the  investigation  of  the  kinetics  of  the  digestive 
enzymes,  the  difficulties  which  beset  all  enzymic  problems  are 
here  supplemented  by  others  due  to  the  complicated  nature  of  the 
proteins.  Observations  made  by  any  of  the  physico-chemical 
methods,  whether  optical  or  electrical,  represent  the  effect  of 
a  large  number  of  decompositions  proceeding  simultaneously, 
and  it  is,  as  the  author  has  repeatedly  emphasised,  surprising 
that  simple  direct  relations  are  so  often  found.  The  processes 
of  decomposition  would  hence  become  decidedly  more  apparent 
if  the  dipeptides,  which  F  i  s  c  h  e  r  's  work  has  rendered 
accessible,  were  employed  as  substrates.  The  first  experiments 
in  this  direction  were  made  by  E  u  1  e  r  (H.,  1907,  51,  213)  on 
glycylglycine,  not  however  with  the  trypsin  of  the  pancreas, 
but  with  the  proteolytic  enzyme  of  the  walls  of  the  intestines, 
the  so-called  erepsin. 

The  course  of  the  reaction  was  followed  by  the  change  in 
conductivity  of  an  alkaline  solution  of  glycylglycine.  The  final 
conductivity,  obtainable  by  complete  hydrolysis  of  the  glycyl- 
glycine, was  determined  beforehand  with  glycine  solutions  of 
corresponding  concentration.  In  the  possibility  of  a  certain 
knowledge  of  this  final  value  seems  to  lie  one  of  the  principal 
advantages  of  using  dipeptides  as  substrates  in  tryptic  and  ereptic 
decompositions.  It  was  also  found  that  the  diminution  of  the 
conductivity  is  proportional  to  the  diminution  of  the  concentra- 
tion of  the  dipeptide.1 

The  first  result  obtained  was  that  the  velocity  of  hydrolysis 
of  glycylglycine  depends  greatly  on  the  alkalinity  of  the  erepsin 
solution.2 

1  It  is  to  be  noted  that  the  existence  of  such  proportionality,  which  forms 
the  basis  of  further  calculations,  was  not  shown  in  previous  investigations 
where  tryptic  or  peptic  digestion  of  protein  was  followed  by  means  of  the 
conductivity. 

2  The  alkalinity  is  expressed  as  the  normality  of  the  total  soda  added. 
The  actual  alkalinity,  that  is,  the  concentration  of  the  free  base,  is  very  much 
smaller,  since  the  greater  part  of  the  soda  is  used  up  in  neutralising  the 
glycylglycine. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS        189 


N/10-glycylglycine.     5  grms.  powdered  erepsin  per  100  c.c. 

Concentration  of  alkali  0  0-04         0-05         0-075 

Reaction  constant X 1000  <0-05        7-0          6-2          2-6 


0-10 
0-2 


Different  preparations  of  the  enzyme  showed  varying  sensitive- 
ness towards  alkali. 

It  was  next  ascertained  that  the  decomposition  of  glycylglycine 
is  a  reaction  of  the  first  order  and  that,  under  favourable  con- 
ditions, the  corresponding  velocity  coefficients  k  remain  constant 
until  the  reaction  is  half  completed.1 

N/10-glycylglycine.     5  grms.  powdered  erepsin  per  100  c.c. 


0-04  N-NaOH. 

b. 
0-05N-NaOH. 

Minutes. 

1000  (a  —x). 

1000/fc. 

Minutes. 

1000(o  -x). 

lOOOfc. 

0 

920 



0 

935 

— 

7 

819 

7-20 

10 

806 

6-46 

15 

721 

7-08 

17 

739 

6-00 

22 

649 

6-88 

25 

654 

6-18 

30 

579 

6-70 

30 

622 

5-90 

In  most  cases  a  considerable  fall  in  the  velocity  occurs  even 
after  about  half  an  hour.  This  depends  principally  on  destruction 
of  the  erepsin,  the  rapidity  of  the  destruction  increasing  with  the 
amount  of  free  alkali  in  the  solution. 

Within  certain  limits  the  velocity  of  reaction  is  almost  independ- 
ent of  the  concentration  of  the  dipeptide.  This  independency 
holds,  however,  only  for  certain  relations  between  the  concen- 
trations of  the  enzyme  and  substrate.  With  small  amounts  of 
enzyme,  the  velocity  of  reaction  rises  with  increase  of  the  con- 
centration of  the  glycylglycine. 


0-1  N-glycylglycine;  0-04  N-NaOH. 
1000/c=0-35 


0 . 2  N-glycylglycine;   0 . 05  N-NaOH. 
1000/0  =  0-55 


Since,  as  has  already  been  mentioned,  the  concentration  of 
free  alkali  has  here  also  a  considerable  influence  on  the  velocity  of 
reaction,  the  influence  of  the  concentration  of  the  substrate  is 
very  difficult  to  explain.  The  author  concludes,  from  the  results 

1  As  the  behaviour  of  erepsin,  like  that  of  other  enzymes,  depends  on  the 
absolute  activity  of  the  solution,  it  is  advisable,  in  order  to  make  differene 
results  comparable,  to  establish  a  standard  action.  A  normal  preparation 
might  be  taken  as  one  which  decomposes  glycylglycine  or  an  optically 
active  dipeptide,  in  5%  solution,  to  the  extent  of  one-half  in  1  hour. 


190 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


of  these  experiments,  that  only  the  alkali  salt  of  the 
d  i  p  e  p  t  i  d  e  undergoes  hydrolysis  (Arkiv  for  Kemi,  1907,  2, 
No.  39). 

As  regards  the  effect  of  the  concentration  of  enzyme,  it  may 
be  said  that,  in  most  of  the  conditions  of  concentration  examined, 
the  velocities  of  reaction  were  proportional  to  the  enzyme-con- 
centrations ;  the  Schiitz-Borissow  law  did  not  hold  in 
any  instance.  The  following  numbers  serve  as  examples: 

Concentration  of  erepsin 5          4          3          2 

1000/c: 6-5      5-4      4-3      2-8 

With  low  concentrations  of  the  enzyme,  especially  with  feeble 
preparations  of  pancreatin,  k  increases  far  more  rapidly  than  the 
concentration  of  the  enzyme. 

With  relatively  high  concentrations  of  enzyme,  deviations  from 
proportionality  in  the  sense  of  S  c  h  ii  t  z's  rule  do,  indeed,  occur; 
but  the  experimental  errors  are  so  great  in  these  reactions,  which 
take  place  in  a  few  minutes,  that  conclusions  cannot  be  drawn 
from  the  results. 

If  optically  active  polypeptides  are  employed  as  substrates, 
the  enzymic  hydrolysis  can  often  be  followed  polarimetrically. 
Such  measurements  have  been  carried  out  byAbderhalden 
and  his  collaborators. 

The  following  results,  obtained  with  d-alanyl-d-alanine, 
indicate  the  time-law  of  this  reaction  (Abderhalden  and 
Koelker,  H.,  1907,  51,  294;  Abderhalden  and 
M  i  c  h  a  e  1  i  s  ,  H.,  1907,  52,  326) : 


0-45  grm.  dipeptide+6  c.c.  pressed  juice. 

0-45  grm.  dipeptide  +4  c.c.  pressed  juice 
+2  c.c.  physiological  salt  solution. 

Min. 

Rotation 

X 

10"z 

k.W*. 

Min. 

Rotation. 

X 

10"z 

&.10*. 

t 

t 

0 

-1-21° 

— 

— 



0 

-1-31° 







3 

-0-96 

0:39 

130 

453 

3 

-I'll 

0-18 

60 

192 

7 

-0-74 

0-59 

84 

390 

7 

-0-98 

0-37 

53 

183 

11 

-0-51 

0-84 

71 

382 

10 

-0-81 

0-54 

54 

202 

18 

-0-20 

1-15 

64 

380 

16 

-0-56 

0-79 

49 

214 

20 

-0-16 

1-19 

59 

373 

25 

-0-21 

1-14 

46 

268 

27 

+0-01 

1-36 

50 

451 

30 

-0-09 

1-26 

42 

294 

34 

+0-05 

1-40 

41 

430 

35 

+0-02 

1-37 

— 

359 

40 

+0-07 

1-42 

35 

— 

43 

+0-07 

1-42 

— 

— 

55 

+0-10 

1-45 

— 

— 

54 

+0-08 

1-43 

— 

— 

65 

+0-10 

1-45 

— 

— 

EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       191 
0-45  grm.  dipeptide+3  c.c.  pressed  juice +3  c.c.  physiological  salt  solution. 


Minutes. 

Rotation. 

X 

IWx 

t 

fc.104 

o-o 

-1-31° 

— 





5-0 

-1-16 

0-19 

380 

125 

6-5 

-1-09 

0-26 

400 

132 

7-5 

-1-05 

0-30 

400 

134 

16-0 

-0-76 

0-59 

369 

142 

22-0 

-0-54 

0-81 

368 

161 

28-0 

-0-32 

1-03 

368 

192 

30-0 

-0-25 

1-10 

314 

209 

38-0 

-0-09 

1-28 

332 

232 

45-0 

+0-01 

1-36 

— 

265 

60-0 

+0-07 

1-42 

~ 

0-6  grm.d-Alanyl-d-alaninein  7  -6  c.c.  pressed 
juice  +0-4  c.c.  physiological  salt  solution. 

0-6  grm.  d-Alanyl-d-alanine  +5-7  c.c.  pressed 
juice  +3  -3  c.c.  physiological  salt  solution. 

Min. 

Rotation 

X 

X 

~t 

&.10* 

Min. 

Rotation. 

X 

X 

T 

fc.10* 

0 
5 
12 
19 
26 
30 

-1-30° 
-1-08 
-0-85 
-0-59 
-0-23 
-0-09 

0 
22 
45 
71 
107 
121 

44 
38 
38 
40 
40 

148 
140 
162 
241 
289 

0 

7 
13 
26 
37 
50 

-1-23° 
-1-09 

-0-91 
-0-46 
-0-11 

0 
21 
39 
84 
119 
140 

30 
30 
32 
32 

28 

60 
92 
144 
216 

40 

30 

It  will  be  seen  that  the  theoretical  formula,  k  =  —  -log  -  — , 

t          a — x 

leads  to  reaction-coefficients  which  show  a  continuous  rise,  the 
amount  of  this  increasing  with  diminution  of  the  amount  of  enzyme 
for  a  constant  amount  of  substrate. 

On  the  other  hand,  with  relatively  small  amounts  of  enzyme, 

x 
the  value  of  the  expression  —  is  strikingly  constant,  and  this 

appears  to  indicate  that  the  change  proceeds  independently  of 
the  amount  of  substrate  still  to  be  decomposed.  But  it  is  more 
probable  that  here,  as  is  always  the  case,  the  velocity  of  reaction, 
dx  :  dt,  is  proportional  to  the  quantity  of  substrate  present,  and 
that  the  reaction,  as  it  proceeds,  is  subjected  to  some  secondary 
acceleration. 


192 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


The  velocity  of  reaction  is  approximately  proportional  to 
the  amount  of  enzyme: 

40  :  7-6  =  5-4, 
30  :  5-7  =  5-4. 

This  proportionality  is  much  less  sharp  with  d-alanylglycine, 
with  which  Abderhalden  and  K  o  e  1  k  e  r  (H.,  1908,  55, 
416)  have  experimented;  the  two  following  tables  appear  on  p. 
422  of  this  paper : 


2  c.c.  Dipeptide  solution  =0-001  normal. 
0  -5  c  c.  pressed  yeast  juice.    4  c.c.  water. 

2  c.c.  Dipeptide  solution  =0-001  normal. 
2-0  c.c.  pressed  yeast  juice.     2-5  c.c.  water. 

Minutes. 

Rotation. 

i 
*.10*. 

Minutes. 

Rotation. 

ft.  10'. 

0 
12 

+0-85°(extrap.) 
0-80 

22 

0 
12 

+0-85°  (extrap.) 
0-70 

70 

31 

0-71 

25 

31 

0-54 

64 

62 

0-63 

22 

62 

0-38 

56 

86 

0-53 

24 

86 

0-28 

56 

118 

0-49 

23 

118 

0-19 

55 

190 

0-36 

20 

— 

— 

— 

Here,  therefore,  the  velocity  of  reaction  increases  considerably 
more  slowly  than  the  concentration  of  the  enzyme,  but  there 
is  no  indication  of  the  validity  of  S  c  h  ii  t  z  's  law. 

With  reference  to  the  effect  of  the  concentration  of  the  sub- 
strate, Abderhalden  andKoelker  (H.,  1908,  54,  363) 
have  made  experiments  with  active  material,  the  following  table 
giving  some  of  their  results : 


(rfr-mol.) 

(2BB-mol.) 

(sfo-mol.) 

3-0  c.c.  d-Alanyl-rf- 
alanine  solution, 
1  -0  c.c.  pressed  yeast 
juice.     2-0  c.c.  water. 

4-0  c.c.  d-Alanyl-d- 
alanine  solution, 
1  -0  c.c.  pressed  yeast 
juice.     1  -0  c.c.  water. 

5-0  c.c.  d-Alanyl-d- 
alanine  solution, 
1  -0  c.c.  pressed  yeast 
juice. 

Minutes. 

Angle. 

Angle. 

Angle. 

4 

-1-36° 

-1-83° 

-2-15° 

12 

-1-31 

-1-75 

-2-10 

32 

-1-17 

-1-58 

-1-97 

61 

-0-95 

-1-31 

-1-73 

92 

-0-67 

-0-97 

-1-43 

128 

-0-42 

-0-69 

-1-16 

167 

-0-18 

-0-38 

-0-86 

190 

-0-08 

-0-23 

-0-69 

238 

+0-03 

+0-01 

-0-35 

308 

+0-05 

+0-13 

+0-02 

357 

+0-05 

+0-12 

+0-15 

377 

+0-11 

+0-18 

+0-29 

EXPERIMENTAL  DATA   OF  ENZYME   REACTIONS       193 

The  dipeptide  employed  in  these  experiments  was,  as  these 
investigators  state,  not  free  from  the  racemic  compound,  so  that 
the  course  of  the  reaction  and  the  calculations  become  more 
complicated.  It  can,  however,  be  seen  from  the  above  numbers 
that  the  amounts  of  dipeptide  hydrolysed  in  a  certain  time  are, 
as  a  first  approximation,  independent  of  the  initial  concentration 
of  the  dipeptide.  The  velocity  constants  calculated  according 
to  the  unimolecular  formula  would,  therefore,  diminish  considerably 
with  increasing  concentration  of  the  dipeptide  instead  of  being 
independent  of  this  concentration,  as  theoretically  they  should 
be.  Within  a  certain  region  of  concentration  or  with  a  certain 
ratio  between  the  concentrations  of  substrate  and  enzyme,  this 
relation  is  found  in  the  case  of  most  enzymes.  It  has  already 
been  shown,  by  experiments  made  by  the  author,  how  greatly 
the  degree  and  character  of  the  reaction  change  with  the  alkalinity 
of  the  solution,  and  confirmation  of  this  result  is  afforded  by  the 
investigations  of  Abderhalden  and  K  o  e  1  k  e  r  (loc.  cit., 
p.  378).  To  obtain  definite  results  in  any  study  of  the  kinetics 
of  trypsin  and  erepsin,  careful  attention  rrust  be  paid  to  the 
alkali-content  of  the  solutions.  If,  as  the  above  measurements 
indicate,  it  is  the  alkali  salts  of  the  dipeptides  or  proteins 
which  undergo  hydrolysis  and  thus  function  as  the  "  active 
molecules,"  the  concentration  of  the  substrate  is  not  merely  that  of 
the  dipeptide  or  protein  but  depends  also  on  that  of  the  alkali 
added;  hence  no  general  simple  relations  for  the  velocity  of 
reaction  will  be  found  if  the  concentration  of  the  dipeptide  or 
protein  alone  is  varied.  It  is  of  more  value  to  ascertain  how 
the  concentration  of  the  alkali  must  be  altered  at  the  same 
time  for  the  theoretical  requirement — independence  of  the 
reaction  constant  on  the  concentration  of  the  substrate — to  be 
fulfilled. 

Abderhalden  and  Koelker  (H.,  1908,  55,  416) 
have  examined  also  the  course  of  the  reaction  in  the  case  of  tri-  and 
tetra-peptides.  Study  of  such  higher  polypeptides  not  only 
serves  to  characterise  the  polypeptides,  with  which  the  velocity 
of  hydrolysis  is  a  characteristic  constant,  but  they  are  also  of 
interest  in  indicating  the  course  of  the  reaction  when,  as  happens 
with  the  true  proteins,  several  hydrolyses  take  place  at  the  same 
time.  As  examples  are  given  the  results  of  two  series  of  exper- 
iments on  d-alanylgly cylglycine ;  since  the  separate  observations 


194 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


are  subject  to  considerable  errors,  the  results  given  here  have 
been  interpolated  graphically. 


3-32  c.c.  Peptide  solution  =0-002  mol. 

4-98  c.c.  Peptide  solution  =0-003  mol. 

1  -0  c.c.  pressed  yeast  juice.     2  •  18  c.c.  H2O. 

1-0  c.c.  pressed  yeast  juice.     0-52  c.c.  HzO. 

Minutes. 

Angle  (interp.) 

k.  10*. 

Minutes. 

Angle  (interp.) 

k.10*. 

0 

1-70° 



0 

2-55° 



20 

1-15 

85 

20 

1-80 

76 

40 

0-70 

96 

40 

1-20 

82 

60 

0-41 

103 

60 

0-80 

83 

80 

0-20 

116 

80 

0-55 

83 

120 

0-26 

82 

It  will  be  seen  that  the  constants  calculated  from  the  unimo- 
lecular  formula  and  given  in  the  third  column,  do  not  increase 
very  greatly  with  the  time. 

A  dynamic  investigation  of  the  vegetable  ereptases 
discovered  by  V  i  n  e  s  (Annals  of  Bot.,  1910,  24,  213),  would  be 
of  considerable  interest. 

At  about  the  same  time  as  the  author,  A  .  E  .  Taylor  car- 
ried out  experiments  on  alkaline  proteolysis,  using  a  chemically 
definite  substrate  in  order  to  render  the  results  more  definite. 
To  this  end  he  prepared  protamine  sulphate  from  the  salmon 
by  K  o  s  s  e  1 '  s  method  and  hydrolysed  it  with  G  r  u  b  1  e  r  '  s 
pancreatin.  Unfortunately  the  results  of  these  researches  are 
given  only  very  briefly  (Berkeley:  On  Fermentation,  1907, 
p.  152).  The  numbers  do,  however,  show  that  the  law  x  =  n\/t 
does  not  hold  for  trypsin.  The  results  of  each  series  of  exper- 
iments can  be  calculated  by  the  formula  for  unimolecular  reac- 
tions: but  the  constants  vary  if  at  different  times  the  concentra- 
tion of  the  substrate  is  altered.  Further,  under  constant  external 
conditions,  the  velocity  of  protamine  digestion  is  simply  and 
directly  proportional  to  the  concentration  of  the  trypsin.  These 
results  were  obtained  with  the  optimal  concentration  of  hydroxyl- 
ions. 


Protamine,  0-100  grm.  in  50  c.c.     Temperature  40C 


Quantity  of  trypsin,  grm. 
Mean  time  of  digestion,  t 
Quantity  of  trypsin  X£ 


0-008  0-006  0-004  0-003  0-002  0-001  0-0005 

37        50     76-5       103       156       313        657 

296      300      306       309      312       313        328 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       195 
Here  also,  then,  the  relation  x  =  KS\/E  does  not  hold. 

In  connection  with  the  above  investigations  on  the  action  of 
proteolytic  enzymes  in  vitro,  reference  may  be  made  to  the 
relations  deduced  by  Arrhenius  (H.,  1909,  63,  323) 
from  the  results  of  the  numerous  experiments  made  by  E .  S  . 
London  on  digestion  in  the  stomach  of  the  dog. 

For  the  experimental  details  reference  must  be  made  to 
London's  original  papers  (H.,  1905,  45,  381;  46,  209;  1906, 
47,  368;  49,  324;  50,  125;  1907,  61,  241,  468;  52,  482;  53,  148, 
240,  246,  326,  334,  356,  403,  429;  54,  80;  1908,  55,  447; 
56,  378-416,  512-553;  57,  113,  529;  1909,  60,  191-283;  61, 
69;  62,  443-464;  1910,  65,  189-218;  68,  346-380).  The  follow- 
ing cases  are  to  be  distinguished: 

1.  Digestion  of  different  quantities  of 
meat  introduced  by  the  mouth.  The  fact,  estab- 
lished by  L  o  n  d  o  n  ,  that  the  amount  digested  in  a  given  time 
is  not  proportional  to  the  quantity  of  nutriment  taken,  is  explained 
as  follows: 

"  The  first  100  grms.  of  meat  lie  close  to  the  stomach-wall 
in  a  layer  of  uniform  thickness.  Within  this  lies  another  layer  of 
100  grms.  of  meat,  this  being  rather  thicker,  as  its  surface  dimin- 
ishes approximately  as  the  cross-section  of  a  cone  as  the  apex 
is  approached.  Further  layers  of  100  grms.  follow,  the  thickness 
continually  increasing.  Into  these  layers  the  gastric  juice  diffuses 
from  the  mucous  membrane.  The  quantity  of  gastric  juice 
in  each  layer  diminishes  considerably  and  the  diminution  increases 
in  extent  as  the  layers  lie  farther  away  from  the  mucous  membrane. 
This  is  conditioned  partly  by  their  increasing  thickness  and  partly 
by  the  laws  of  diffusion,  which  require  such  increasing  diminution 
even  for  layers  of  equal  thickness.  Hence  it  comes  about  that 
the  innermost  layers  are  not  perceptibly  digested  and  the  amount 
digested  in  a  given  time  increases,  as  the  amount  of  food  taken 
is  augmented,  only  to  a  maximum." 

As  far  as  digestion  is  concerned,  every  layer  is  independent 
on  every  other  and  the  total  quantity  digested  is  the  sum  of  those 
digested  in  the  various  layers.  On  the  simple  assumption  that 
the  digestion  in  the  outermost  layer  is  always  the  same,  the 
jmmber  of  layers  which  may  lie  inside  has  been  calculated  by 
Arrhenius  from  London's  results  by  means  of  an 


196 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


empirical  formula.  The  latter  gives  the  quantities  of  food 
digested  in  a  certain  time  in  the  various  layers  and  corresponds 
very  well  with  the  experimental  figures. 

With  the  aid  of  this  formula  Arrhenius  has  prepared  a 
table  showing  the  time-course  of  the  digestion  which  he  represents 
also  graphically  (Fig.  5).1 


icoo 


DIGESTION  OF  DIFFERENT  QUANTITIES 
OF  MEAT,   100-1000  GRAMS. 


\ 


400 


200 


\\ 


\ 


10         11 


1  From  C  h  i  g  i  n  and  Lobassow's  results  (Dissertation,  St. 
Petersburg,  1896),  H  e  r  z  o  g  (Zeitschr.  f.  allg.  Physiol.,  1904,  4,  163)  has 
attempted  to  calculate  the  time-course  of  the  secretion  of  gastric  juice. 

Determinations  were  made  of  the  quantities  of  pepsin  (measured  by  the 
number  of  c.c.  of  gastric  juice  and  its  specific  digestive  power)  secreted  in  a 
blind  sac  in  the  fundus  part  of  the  stomach,  when  food  was  introduced  into 
the  stomach.  According  to  Herzog's  calculations,  the  secretion  of 
pepsin  follows  the  formula  for  unimolecular  reactions. 

With  introduction  of  solid  food  the  values  of  k  obtained  are  moderately 
constant.  But  in  what  degree  the  measurement  and  calculation  of  the 
quantities  of  pepsin  by  the  Russian  workers  are  free  from  objection,  cannot 
well  be  judged.  C  h  i  g  i  n  and  Lobassow's  results  have  recently 
been  referred  to  in  a  paper  by  Arrhenius  (H.,  1909,  63,  323). 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       197 

From  the  curve  for  1000  grms.  the  quantities  remaining 
undigested  after  a  certain  number  of  hours  have  been  calculated, 
the  results  being  as  follows: 

Time  in  hours:  0      1      2      3.     4      5      6      7      8      9101111-5 

Amount  undigested:  1000  875  750  627  507  390  276  180  100  51  24     7     3 
Difference  per  hour:  125  125  123  120  117  114    96     80  49  27  17 

It  will  therefore  be  seen  that  the  quan- 
tity digested  is  at  first  very  nearly  pro- 
portional to  the  time,  but  gradually  di- 
minishes later. 

This  apparent  contradiction  ofSchiitz's  rule  depends  on 
the  fact  that  in  vivo  the  products  of  digestion  are  continually 
removed,  whilst  in  vitro  they  remain  in  the  solution  and 
hence  retard  the  reaction. 

The  above  table  leads  to  a  very  important  rule.  It  will 
be  seen  that  for  practically  complete  digestion  of  1000  grms.  of 
meat  11-5  hours  are  necessary.  The  time  t  required  to  digest 
another  quantity  M  is  calculated  by  subtracting  from  11-5  hours 
the  time  taken  to  digest  (1000-M)  grms.  of  meat  when  the 
initial  quantity  is  1000  grms.  In  this  way  the  times  £0bs.  given 
below  for  varying  values  of  M  have  been  obtained. 


M 

W 

kale. 

(kbs.  —  kale.) 

1000 

11-50 

10-81 

+0-69 

900 

10-70 

10-26 

-fO-44 

800 

9-90 

9-67 

+0-23 

700 

9-09 

9-05 

+0-04 

600 

8-27 

8-38 

-0-11 

500 

7-44 

7-65 

-0-21 

400 

6-60 

6-84 

-0-24 

300 

5-73 

5-92 

-0-19 

200 

4-74 

4-84 

-0-10 

100 

3-45 

3-42 

+0-03 

50 

2-40 

2-42 

-0-02 

25 

1-55 

1-71 

-0-16 

Besides  the  values  of  £obs.,  those  of  £caic.  are  also  given,  these 
being  calculated  from  the  formula 


198  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Hence,  the  time  required  for  the  virtually  complete  digestion  of 
flesh-food  introduced  per  o  s  is  very  nearly  proportional  to  the 
square-root  of  the  quantity  of  food. 

This  square-root  law  is  repeated  in  numerous  series  of  experi- 
ments and  also  with  different  nutriment.  It  holds 
not  only  for  ordinary  meat  but,  as  was  shown  byArrhenius, 
for  the  digestion  of  gliadin  (London,  H.,  1908,  56,  394), 
of  the  albumin  of  hens'  eggs  (London,  H.,  1908,  56,  405) 
and  of  bread  (London,  H.,  1906,  49,  359),  and  is  therefore 
of  the  greatest  importance  for  calculating  the  digestion  periods 
of  solid  foods. 

2.  Digestion  of  meat  introduced  directly 
into  the  stomach  by  a  fistula.  If  the  nutriment 
is  taken  per  o  s  ,  a  reflex  secretion  of  gastric  juice  occurs,  so 
that  the  digestion  is  greatly  accelerated.  When,  however,  the 
introduction  of  food  takes  place  through  a  fistula,  the  secretion 
of  gastric  juice  produced  by  the  stimulating  action  of  the  meat 
on  the  stomach-wall  is  at  first  gradual  and  increases  with  the  time; 
indeed,  as  the  following  calculation  shows,  the  quantity  of  gastric 
juice  in  the  stomach  is  about  proportional  to  the  time  elapsing 
since  the  introduction  of  the  meat. 

Thus,  if  the  digestion  is  proportional  to  the  quantity  of  gastric 
juice  present  and  the  latter  to  the  time,  the  amount  digested  per 

unit  of  time,  — ,  is  proportional  to  the  time  t  and  to  the  quantity 
at 

of  meat  (M  —  x)  still  present  (M  being  the  amount  initially 
added),  or,  mathematically: 


dt 
from  which  follows: 


London  and  Pewsner's  results,  given  below,  have  been 
calculated  according  to  this  formula. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       199 


A.  Feeding  of  dog  per  fistulam.  Eyes  and  nose  covered. 


t  (hours) 

Undigested 
quantity  (M—x) 

(M-x)Caic. 

,     Difference. 

0 

100 

100 

— 

2 

84 

84-7 

-0-7 

4 

56 

51-5 

+4-5 

6 

20 

22-5 

-2-5 

8 

7 

7-0 

0 

9 

0 

3-5 

-3-5 

B.  Feeding  per  fistulam.     Eyes  and   nose  uncovered. 


t  (hours). 

Undigested 

quantity  (M—x). 

(M-a)caic. 

Difference. 

0 

100 

100 



2 

84 

83-7 

+0-3 

4 

53 

49-1 

+3-9 

6 

18 

20-1 

-2-1 

8 

5 

5-8 

-0-8 

9 

0 

2-7 

-2-7 

In  the  experiment  A,  the  constant  had  the  value  k  =  0-0180 
and  in  B,  k  =  0-0193. 

3.  If  we  now  ask  how  the  total  course  of  the  digestion 
is  to  be  represented  (i.e.,  not  only  as  regards  the  time  necessary 
for  local  digestion),  it  may  in  general  be  stated  that,  in  so  far  as 
small  quantities  of  food  (the  layer  which,  according  to  the  above 
assumption,  is  in  immediate  contact  with  the  stomach-wall) 
are  concerned,  digestion  follows  the  law  holding  for  unimolecular 
reactions.  This  is  shown  by  the  following  examples : 


Digestion  of  boiled  protein,  200  grma. 


Digestion  of  meat,  200  grms. 


t 

(100  -z) 

(100-aOcalc. 

Diff. 

t 

(100-3) 

(lOO-z)calc. 

Diff. 

0 

100 

100 



0 

100 

100 



1 

70 

65 

+5 

1 

60 

56 

+4 

2 

32 

42 

-10 

2 

31 

32 

-1 

3 

28 

27 

+  1 

3 

15 

18 

-3 

4 

18 

18 

0 

4 

5 

10 

-5 

5 

15 

12 

+3 

5 

0 

6 

—6 

6 

4 

7 

-3 

(7 

0 

5 

-5) 

200  GENERAL   CHEMISTRY  OF  THE  ENZYMES 

• 

By  (a  —  x)  is  denoted  the  quantity  of  undigested  protein  still 
in  the  stomach  at  the  time  t.  The  calculated  figures  are  obtained 
by  means  of  the  formula: 

7      1  100 

fc=-7-log 


t      &  100-ar 

The  same  formula  holds  for  the  digestion  of  dissolved 
carbohydrates  (amylodextrin)  . 

4.  The  resorplioii  of  a  sugar  solution  in  the  intestines  also  follows 
the  unimolecular  law. 

If  a  solution  of  glucose  is  introduced  into  the  intestine,  the  latter 
gives  up  water  to  it  if  the  solution  is  concentrated,  but  takes  up 
water  from  a  dilute  solution.  The  quantity  of  water  yielded  to  the 
solution  reaches  a  maximum,  amounting  to  900  c.c.,  corresponding  with 
the  capacity  of  the  body  to  give  up  water  From  London's  results, 
Arrhenius  has  calculated  the  concentration  of  the  solution  when 
no  water  is  taken  up  o  •  give  out  by  the  intestine,  this  being  10  •  5% 
of  glucose.  As  was  to  be  expected,  it  is  found  that  the  amount  of  water 
taken  from  the  intestine  is  proportional  to  the  excess  of  the  concentra- 
tion over  10-5%.  The  law  followed  is  hence  expressed  by  the  equation: 


where  W  is  the  amount  of  water  given  up  by  the  intestine  and  (C  —  10-5) 
represents  the  excess  of  the  concentration  over  10  •  5%. 

RENNET    (CHYMOSIN) 

By  the  action  of  rennet,  casein  is  converted  into  paracasein 
(compare  p.  45);  whether  at  the  same  time  some  component, 
possibly  an  albumose,  is  split  from  the  casein  molecule  is  a  dis- 
puted question.  On  account  of  this  supposed  partial  hydrolysis, 
and  of  the  occurrence  of  pepsin  and  chymosin  together,  the  latter 
is  classed  with  the  true  proteolytic  enzymes. 

It  must  here  be  borne  in  mind  that  the  clotting  of  milk  by 
rennet  consists  of  two  processes  (Hammarsten)  :  chymosin 
only  accelerates  the  conversion  of  casein  into  paracasein,  a 
change  which  proceeds  without  calcium  salts.  These  salts  are 
necessary  only  for  the  precipitation  of  the  curd  (para- 
casein) for  which,  indeed,  no  rennet  is  required. 

It  would  therefore  be  expected  that  the  time  which  clotting 
takes  to  begin  is  made  up  of  two  periods:  (1),  that  necessary  for 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       201 


the  casein  to  be  transformed,  almost  completely,  into  paracasein — 
time  of  conversion,  and  (2),  that  necessary  for  the  formation 
of  a  visible  clot — time  of  separation.  Such  an  assumption  was 
advanced  by  Fuld  (Hofm.  Beitr.,  1902,  2,  169).  According 
to  this  author,  the  time  of  separation  amounts  to  several  days 
or  to  a  few  minutes  or  even  less,  in  dependence  on  the  temperature 
of  the  paracasein  solution,  and  is  so  inappreciable  in  comparison 
with  the  long  period  of  conversion  of  the  experiments  referred 
to  above,  that  it  may  be  unhesitatingly  neglected.  On  the  other 
hand,  R  e  i  c  h  e  1  and  S  p  i  r  o  ,  in  their  most  recent  investiga- 
tion, regard  such  an  assumption  as  that  of  F  u  1  d  as  unjustified, 
and  express  the  view  that,  with  reference  to  its  time-course,  the 
whole  clotting  process  must  be  considered  as  a  single  process 
(Hofm.  Beitr.,  1906,  8,  15). 

According  to  Bang  (Skand.  Arch.  f.  Physiol.,  1911,  25, 105), 
the  clotting  of  milk  by  rennet  is  an  extremely  complicated  process, 
in  which  the  calcium  salts  present  are  distributed  between  organic 
acids,  lactalbumin  and  lactoglobulin,  whilst,  on  the  other  hand,  the 
casein  is  distributed  among  the  basic  constituents  of  the  solution. 

The  clotting  process  obeys  the  law,  [E].t  =  const.,  or 

The  product  of  the  enzyme-concentra- 
tion [#]  a  n  d  time  of  clotting  t  is  constant. 
This  relation  was  first  observed  by  Segelcke  and  S  t  o  r  c  h 
(Ugeskrift  for  Landmaend,  1870)  and  was  subsequently  fully 
confirmed  by  H  a  n  s  e  n  and  S  o  x  h  1  e  t  (Milchzeitung,  1877). 
The  most  complete  investigations  of  the  action  of  rennet  are 
due  to  F  u  1  d  and  to  R  e  i  c  h  e  1  and  S  p  i  r  o  . 

At  40°,  E  .  F  u  1  d  (Hofm.  Beitr.,  1902,  2,  172)  obtained  the 
following  numbers,  which  proved  the  exact  and  general  validity 
of  the  time-law  even  with  small  amounts  of  rennet: 


Quantity  of  rennet. 

Time  of  clotting,  t,  in  seconds. 

Product. 

0-4 

6 

24 

0-4 

6-6 

26 

0-2 

13 

26 

0-1 

25 

25 

0-8 

6 

48 

0-4 

17 

44 

0-2 

22 

44 

0-1 

45 

45 

202 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


According  to  C.  G  e  r  b  e  r  (Soc.  BioL,  1907,  63,  575),  who  has 
recently  made  a  thorough  study  of  the  clotting  process,  it  is  essen- 
tial, when  working  with  the  rennet  of  commercial  pepsin,  to 
employ  temperatures  between  25  and  30°  so  that  the  enzyme 
shall  be  under  normal  conditions.  Within  these  limits  of  tem- 
perature, G  e  r  b  e  r  finds  that  the  law  of  Segelcke  arid 
S  t  o  r  c  h  holds  closely  for  parachymosin. 

Drops  of  parachymosin  #....1  2  3  46  7          8          9      10 

Time  of  clotting,  t 29-66'  14-75'  10-30'  7-60'  5-50'  4-50'  3-66'  3-16'  2-92' 

E.t 29-6      29-5         31     30-3       33    31-5    29-3    28-4    29-2 

M  a  d  s  e  n  has  also  investigated  the  duration  of  milk-clotting, 
the  method  used  being  similar  to  that  employed  with  pepsin 
action  (see  Arrhenius,  Immunochemistry,  p.  72). 

He  adds,  for  instance,  varying  quantities  of  rennet  to  equal  amounts 
of  milk  in  test-tubes,  places  the  mixtures  in  a  water-bath  at  a  definite 
temperature,  takes  them  out  after  time  t,  cools  them  quickly  and  deter- 
mines the  smallest  quantity  of  rennet  L  which  has  produced  clotting. 

COAGULATION  OF  MILK  BY  RENNET  SOLUTIONS  OF  DIFFERENT  CONCENTRATIONS 

AT  36-55° 


Minutes. 

L. 

Lt. 

Minutes. 

L. 

Lt. 

4 

0-08 

0-32 

35 

0-007 

0-25 

6 

0-05 

0-30 

50 

0-005 

0-25 

9 

0-033 

0-30 

70 

0-004 

0-28 

11 

0-024 

0-26 

80 

0-0032 

0-26 

12 

0-019 

0-23 

100 

0-0028 

0-28 

14 

0-0175 

0-25 

120 

0-0025 

0-30 

20 

0-013 

0-26 

180 

0-00185 

0-33 

25 

0-010 

0-25 

240 

0-0017 

0-41 

30 

0-007 

0-21 

Bang  (Pflug.  Arch.,  1900,  79,  425)  found  that  the  relation, 
[E]  t  =  const.,  did  not  hold  for  parachymosin. 

By  a  series  of  careful  experiments  R  e  i  c  h  e  1  and  S  p  i  r  o 
(Hofm.  Beitr.,  1905,  7,  485)  showed  that  the  relation  between 
quantity  of  rennet  L  and  period  of  clotting  t  can  be  expressed 
generally  by  the  formula: 


Ln.t  =  const. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS   203 


The  exponent  n  of  L  changes  with  the  nature  and  amount 
of  the  other  substances  in  the  solution,  and  in  a  liquid  of  the  com- 
position of  milk  it  assumes  exactly  the  value  1,  so  that  the  law 
holds  in  its  simplest  form, 

L.t  =  const. 

In  the  following  experiments  the  solutions  were  prepared  by 
dilution  with  whey,  the  clotting  action  of  which  was  negligible 
in  comparison  with  that  of  the  enzyme  added. 


Milk. 

c.c.  of  35% 
rennet  diluted 
with  whey. 

Whey. 

Time  of  clotting  t  in  seconds. 

Whey  I. 

Whey  II. 

Whey  III. 

0-5 

0-5 

4-0 

22 

22 

33 

1-0 

0-5 

3-5 

17 

22-5 

27 

1-5 

0-5 

3-0 

18 

27 

28 

2-0 

0-5 

2-5 

17 

25-5 

26-5 

2-5 

0-5 

2-0 

18 

24-5 

25-5 

3-0 

0-5 

1-5 

16 

26 

26 

3-5 

0-5 

1-0 

16 

26 

24 

4-0 

0-5 

0-5 

15 

25 

26 

4-5 

0-5 

~ 

16 

24 

27 

As  will  be  seen  from  these  results,  the  constant  Lt  or,  since 
L  is  the  same  in  all  cases,  the  time  of  clotting,  is  independent 
of  the  concentration  of  milk  (casein)  in  the 
solution  from  20%  up  to  90  %. 

Replacement  of  the  whey  by  0-9%  sodium  chloride  solution 
resulted  in  constancy  of  the  time  of  clotting  for  more  dilute  as 
well  as  for  more  concentrated  solutions. 

More  extended  investigations  then  showed  that  the  difference 
between  the  times  of  clotting  for  dilute  and  concentrated  milks 
was  approximately  proportional  to  the  difference  between  the 
dilutions.  Hence,  if  V  denotes  the  volume  of  the  diluted  milk, 
M  that  of  the  undiluted  milk,  and  t  and  t'  the  times  of  clotting 
in  the  two  media,  then: 


M 


V-M 


const. 


204 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


This  is  shown  by  an  extract  from  Table  VII  of  R  e  i  c  h  e  1 
and  S  p  i  r  o  '  s  paper. 


Rennet. 

Milk. 

Whey. 

Time. 

«-<'>  (T^T) 

1-0 

0-2 

8-8 

110 

1-60 

1-0 

0-6 

8-4 

50 

1-81 

1-0 

1-0 

8-0 

39 

1-93 

1-0 

2-0 

7-0 

28-6 

1-75 

1-0 

4-0 

5-0 

24 

1-60 

1-0 

8-0 

1-0 

22 

1-60 

The  influence  of  the  Ca-ions  is  also  expressed  by  a  simple  and 
important  relation, 


Time  t  for  amount  of  rennet. 

Constant,  Ca"  Xt  for  amount  of 

Milk, 

CaClj, 

0  /    _ 

rennet. 

c.c. 

u/oo 

1-0 

0-5 

0-25 

1-0 

0-5 

0-25 

8-0 

— 

95 

48 

24 

57-0 

28-8 

14-4 

8-0 

0-05 

88-6 

45-6 

23 

57-6 

29-6 

15-0 

8-0 

0-1 

79 

41-6 

22 

55-3 

29-1 

15-4 

8-0 

0-2 

66-4 

36 

19 

53-1 

28-8 

15-2 

8-0 

0-5 

48 

26-4 

14 

52-8 

29-0 

15-4 

8-0 

1-0 

30 

18-2 

10-6 

48-0 

29-1 

17-0 

8-0 

2-0 

17 

11 

6-8 

44-2 

28-6 

17-7 

8-0 

5-0 

10 

7-4 

5-4 

56-0 

41-4 

30-2 

8-0 

10-0 

13 

9-2 

6-2 

137-8 

97-5 

65-7 

8-0 

20-0 

22 

15 

8-6 

453-2 

309-0 

177-2 

The  values  of  the  constant,  which  show  little  change  up  to  about 
0-02%  of  calcium,  are  calculated  on  the  assumption  that  the  Ca-ions 
in  the  milk  amount  to  0-6%o;  about  15%  of  the  total  calcium  of 
the  milk  must  then  be  in  an  ionised  condition. 

Further,  as  was  shown  in  a  subsequent  paper  (Hofm.  Beitr., 
1906,  8,  15)  the  rennet-action  a  is,  in  general  (not  merely  at  the 
clotting  point),  directly  proportional  to  the  quantity  of  enzyme 
L  and  to  the  time  t,  that  is 


=  L.t.  const. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS        205 

The  more  recent  assertion  of  H.Kottlitz  (Arch,  intern,  de 
Physiol.,  1907,  5,  140)  that  S  c  h  ii  t  z  '  s  rule  holds  also  for  rennetic 
action  in  no  way  diminishes  the  importance  of  R  e  i  c  h  e  1  and  S  p  i  r  o  '  s 
results. 

Several  attempts  have  been  made  to  estimate  the  value  of  the 
time-laws  determined,  on  the  one  hand,  for  the  action  of  rennet 
and,  on  the  other,  for  that  of  pepsin,  as  criteria  for  the  disputed 
identity  of  chymosin  and  pepsin. 

The  discussion  of  this  question  would  not  be  in  place  in  a 
chapter  dealing  with  the  chemical  dynamics  of  the  enzymes; 
but  it  may  at  least  be  asserted  that,  especially  since  H  a  m  - 
marsten's  comprehensive  investigation  (H.,  1908,  56,  18), 
it  is  no  longer  possible  to  regard  the  clotting  of  milk  simply  as  a 
peptic  action.  The  influence  of  concentration,  and  also  that  of 
temperature,  are  so  divergent  in  the  cases  of  clotting  and  diges- 
tion, that  it  must  at  the  least  be  assumed  that  two  different 
enzymic  groups  are  united  in  one  molecule.  This  assumption, 
so  long  as  the  preparation  of  the  enzymes  in  a  pure  state  is  not 
achieved,  can  be  neither  refuted  nor  proved,  and  is  indeed,  under 
present  circumstances,  of  subordinate  importance. 

FIBRIN-FERMENT 

E  .  F  u  1  d  (Hofm.  Beitr.,  1902,  2,  514)  mixed  the  plasma  of 
bird-blood  with  the  enzyme  solution  (obtained  by  extracting 
muscle  with  0-8%  sodium  chloride  solution).  The  velocity 
of  clotting  increased  more  slowly  than  the  concentration  of  the 
enzyme,  the  results  agreeing  approximately  with  S  c  h  ii  t  z  '  s 
rule  but  more  accurately  with  the  expression : 

^  \  0-585 

aj     ' 

where  vi  and  v%  are  the  velocities  of  clotting  and  E\  and  E%  the 
concentrations  of  the  enzyme.  For  protracted  clotting  periods, 
that  is,  for  low  enzyme-concentrations,  this  relation  fails,  the 
duration  of  clotting  then  showing  disproportionate  increase. 

It  has  been  shown,  especially  by  M  a  r  t  i  n  (Journ.  of  Physiol., 
1905,  32,  207),  that  the  rule  obeyed  by  chymosin  and  numerous 
other  enzymes — that  the  same  amount  of  action  occurs  for  equal 


206 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


values  of  the  product  E.t,  no  matter  what  the  values  of  E  and 
t — holds  also  for  the  fibrin-ferment. 


ZYMASE 

Although  the  "  zymase  "  of  pressed  yeast-juice  is  accompanied 
by  an  extremely  large  quantity  of  various  other  substances  and 
is  further  removed  from  a  state  of  purity  than  is  the  case  with 
any  other  enzyme,  yet  in  recent  years,  especially  by  the  work  of 
Harden  and  Young,  important  information  has  been 
obtained  concerning  the  chemistry  of  alcoholic  fermentation. 

If  the  final  products,  alcohol  and  carbon  dioxide,  of  pressed 
yeast-juice  containing  sugar  are  studied  quantitatively,  as  has 
been  done  by  the  author  (H.,  1905,  44,  53),  the  following 
results  are  obtained: 

The    expression    —  •  log  —  —  =•=  k    gives    moderately    constant 


numbers  during  the  first  half  of  the  reaction,  but  subsequently  the 
value  of  k  exhibits  considerable  diminution.  To  this  effect,  two 
principal  causes  contribute,  firstly,  the  separation  of  protein 
substances  which  carry  down  part  of  the  fermentation  enzymes, 
and  secondly,  alteration  of  one  of  the  activators  of  the  zymase; 
this  activator — consisting  of  organic  compounds  of  phosphoric 
acid — is  attacked  by  the  lipase  of  the  yeast-juice,  the  latter 
gradually  becoming  impoverished  in  the  "  co-enzyme  "  which 
is  of  s\ich  great  importance  to  alcoholic  fermentation. 

The  quantity  of  carbon  dioxide  evolved  per  unit  of  time  was 
determined  partly  by  the  loss  in  weight  of  the  solution  and  partly 
volumetrically. 

4  grms.  glucose  in  20  c.c.  of  yeast-juice. 


Minutes. 

x  (grms.  CO2). 

(a—x). 

fc.104. 

0 



1-800 



80 

0-078 

•722 

2-4 

315 

0-299 

•501 

2-51 

379 

0-360 

•440 

2-56 

505 

0-460 

•340 

2-54 

1024 

0-779 

•021 

2-41 

1180 

0-810 

0-990 

(2-20) 

1544 

0-899 

0-901 

(1-95) 

2119 

0-955 

0-845 

(1-55) 

EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       207 


For  about  18  hours,  the  velocity  of  reaction  is  here  moderately 
constant.  This  is,  however,  a  favourable  case  and  under  other 
conditions,  as  is  shown  by  the  following  table,  the  pressed  juice 
remains  unchanged  only  for  6-8  hours. 

The  influence  of  the  concentration  of  the  substrate  is  also 
shown  by  the  following  results. 

20  c.c.  juice +8  grms.  of  glucose  in  20  c.c.  of  solution. 


Minutes.   :  x(grms.) 

(o-*). 

k.  10*. 

Minutes. 

x(grms.) 

(a-x). 

fc.105. 

3-909 

3-909 

0 

3-900 

— 

0 

3-900 

— 

161 

0-078 

3-822 

5-44 

160 

0-0815 

3-8185 

5-38 

260 

0-120 

3-780 

5-24 

257 

0-1195 

3-7805 

5-24 

358 

0-161 

3-739 

5-11 

355 

0-159 

3-741 

5-05 

404 

0-181 

3-719 

5-10 

404 

0-180 

3-720 

5-10 

5-21 

5-19 

20  c.c.  juice +2  grms.  of  glucose  in  20  c.c.  of  solution. 


Minutes. 

x(grms.) 

(a-x). 

fc.10*. 

Minutes. 

x(grms.) 

(a-x). 

k.lW. 

0-977 

0-977 

0 

0 

0-968 

— 

0 

0 

0-962 

— 

167 

0-0855 

0-8825 

2-40 

153 

0-081 

0-881 

2-50 

240 

0-123 

0-845 

2-46 

260 

0-123 

0-839 

2-27 

332 

0-152 

0-816 

2-29 

331 

0-182 

0-780 

2-75 

2-38 

2-51 

In  addition  to  these  series  of  experiments,  three  others  were 
carried  out,  also  with  concentrations  of  sugar  in  the  ratio  4:1. 
The  constants  obtained  were  as  follows: 


i 

fci  .  105 

:*,. 

105 

No. 

4. 

5-2 

:  24 

•4 

=   1 

:  4 

•  7 

11 

5. 

2-5 

:  12 

0 

=   1 

:  4 

•8 

11 

7. 

15-0 

:  75 

•0 

=   1 

:  5 

•0 

" 

6. 

20-0 

:  97 

1 

:  4 

•85 

It  will  be  seen  that  the  velocities  are  not,  as  they  should  be 
according  to  theory,  independent  of  the  concentration  of  the 
substrate;  also  they  are  not  inversely  proportional  to  the  initial 


208  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

concentration  a,  so  that  ka  is  not  a  constant  magnitude  but,  in 
the  region  of  concentration  investigated,  increases  as  a  decreases. 
In  each  of  the  four  series  of  experiments,  ai  :  a2  =  4  :  1. 

ftiai.106  '    &2a2.106 

No.  4.        208  244 

"    5.        100  120 

"    7.        600  750 

"    6.        800  990 

Whilst,  in  general,  the  velocity  of  reaction  increases  either 
proportionally  to,  or  slower  than,  the  concentration  of  enzyme, 
the  velocity  of  fermentation  increases  more  rapidly  than  the 
concentration  of  the  pressed  juice  but  always  slower  than  its 
square. 

If  the  exponents  n  are  calculated  according  to  the  equation 

In  k\  —  In  k% 


1 7 > 

In  ci  —  ln  €2 

it  is  found  that,  for  a  constant  sugar-content,  n  increases  with 
diminution  of  k,  that  is,  with  diminution  of  the  concentration 
of  the  zymase: 

ki .  106  n 

100  1-29 

86  1-33 

35  1-52 

12  1-67 

The  numbers  appear  to  indicate  that,  with  very  high  fer- 
mentative activity,  proportionality  between  the  concentration 
of  the  pressed  juice  and  the  velocity  of  fermentation  is  attained. 

Finally,  if  pressed  juice  containing  sugar  is  diluted,  that  is, 
the  volume  increased  while  the  absolute  amounts  of  the  juice  and 
sugar  remain  constant,  the  following  relations  are  found: 

f  Concentration"32  :    52  =  1:1-63          f    20:   30:    50  =  1:1-5  :2-5 
)    [Velocity          192:282=1:1-47  ()  {  120  : 188  :  315  =  1:  1-57:  2-63 

The  mean  of  these  two  experiments  indicates,  within  the  limits 
of  concentration  employed,  approximate  proportionality  between 
concentration  and  velocity. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       209 


Prior  to  these  experiments  with  pressed  yeast-juice,  A  b  e  r  s  o  n 
Rec.  Trav.  Chim.  Pays-Bas,  1903,  22,  78)  had  studied  the  course 
of  the  alcoholic  fermentation  of  glucose  by  living  yeast-cells. 

It  must  however  be  emphasised  that  Aberson's  observa- 
tions were  made  polarimetrically,  so  that  he  measured  the  amount 
of  sugar  disappearing  during  the  reaction.  But  new  measurements 
made  by  E  u  1  e  r  and  Johansson  (H.,  1912,  76,  347)  with 
living  yeast  show  that  the  diminution  in  rotation  of  a  fermenting 
sugar  solution  is  by  no  means  proportional  to  the  evolution  of 
carbon  dioxide.  This  is  indicated  by  the  following  numbers: 


CO2  evolved. 

Change  in  rotation. 

Concen-i  ^ 

Dif- 

No. 

Temp. 

tration 
of 

Sugar. 

yeast  in 
25  c.c. 
solution 

Time. 
Min. 

Grms. 

Per 
cent. 
A 

Degrees. 

Per 
cent. 
B 

fer- 
ence. 
B-A 

(1 

30° 

10% 

0-5 

125 

0-0936 

7-7 

5-37-4-67=0-70 

13  1 

5-4 

12 

30 

10 

0-5 

181 

0-1492 

12-2 

5-32-4-46  =  0-86 

16  2 

4-0 

3 

30 

10 

1 

42 

0-0644 

5-3 

5-33-4-80=0-53 

10-0 

4-7 

4 

30 

10 

1 

63 

0-1160 

9-5 

5-33-4-48  =  0-85 

16-0 

6-5 

5 

30 

10 

1 

232 

0-4918 

40-3 

5-33-2-84  =  2-49 

47-0 

6-7 

6 

30 

10 

1 

406 

0-7256 

59-4 

5-33-1-85  =  3-48 

65-9 

6-5 

The  cause  of  this  difference  has  not  been  fully  elucidated. 

Owing  to  this  circumstance,  Aberson's  experiments 
give  information  concerning  only  the  first  phase  of  alcoholic 
fermentation,  namely,  the  transformation  of  the  sugar.  The 
results  of  his  numerous  experiments  do  not  correspond  with  the 
expected  law,  but  the  values  given  in  the  last  column  of  the  fol- 
lowing table — which  is  taken  from  that  given  on  p.  97  (1  o  c  . 
c  i  t . ) — are  far  more  nearly  constant. 


Minutes. 

Rotation  of 
the  glucose 
solution. 

1    .         a 
fc=      log  

t          a—x 

1         a+x 

KH        •  log 
t          a—x 

0 

34-1 

— 

— 

Temperature,  29-3°. 

31 

33-0 

45-9 

90-0 

Volume,  600  c.c. 

91 

30-9 

47-0 

90-0 

Amount  of  yeast  : 

125 

29-7 

48-0 

90-0 

0  mins.  :  0-288grm. 

213 

26-7 

50-6 

90-0 

dry  yeast  per  50  c.c. 

306 

23-5 

51-2 

91-0 

514  mins.  ':  0-294 

393 

20-9 

54-1 

90-0 

grm.   dry  yeast  per 

514 

17-7 

55-4 

90-3 

50  c.c. 

210 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


In  the  fermentation  of  similar  sugar  solutions  with  varying 
quantities  of  yeast,  A  b  e  r  s  o  n  obtained  proportionality  between 
the  velocity  of  reaction  and  the  amount  of  yeast  (1  o  c  .  c  i  t ., 
p.  84): 


Grms.  yeast 
k 


60 

271 


20 
93-4 


Grms.  yeast 
k 


25 
165 


15 

104 


SI  at  or  (Journ.  Chem.  Soc.,  1906,  89,  128),  who  measured 
volumetrically  the  carbon  dioxide  evolved,  also  found  propor- 
tionality between  the  velocity  of  fermentation  and  the  quantity 
of  yeast  added.  He  showed  likewise  that  the  initial  velocity 
of  fermentation  is  independent — except  with  very  dilute  solu- 
tions—  of  the  sugar-content. 

The  fermentation  experiments  of  H  e  r  z  o  g  (H.,  1902,  37, 
149)  and  G  r  i  g  o  r  i  e  w  (H.,  1904,  42,  299)  also  refer  to  hetero- 
geneous systems. 

The  course  of  fermentation  with  permanent  yeast  (which, 
however,  contained  glycogen)  is  shown  by  the  following  table  from 
H  e  r  z  o  g  '  s  paper. 

Concentration,  1-136  grms.  glucose  (a  =  l)  and  1-2  grms.  zymase  (perma- 
nent yeast)  in  100  c.c.  Temperature  24-5°. 


t 

a—  x 

106            a 

106         a+x 

.   '*°8 

t           a—x 

.   '*°S 

t           a—x 

120 

0-961 

144 

141 

240 

0-931 

129 

133 

1200 

0-687 

137 

117 

1440 

0-627 

140 

118 

1740 

0-560 

145 

117 

2690 

0-403 

146 

111 

3000 

0-365 

146 

108 

4140 

0-271 

137 

99 

The  exponent  n  of  the  enzyme-concentration  which  shows 
the  increase  of  the  velocity  of  the  reaction,  is  greater  in  the  exper- 
iments with  permanent  yeast  than  in  those  with  the  pressed 
juice.  The  following  table  indicates  the  behaviour  of  living 
yeast,  acetone-permanent  yeast  and  yeast-juice  from  a  dynamical 
point  of  view. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS      211 


Reaction  constant. 

Living  yeast. 

Acetone  permanent  yeast, 

Zymase  solution 
(pressed  juice). 

k  = 

Influence    of    the 
amount  of  yeast 
or  concentration 
of  zymase 
log  ki  —  log  kz 

1         a+x 

1  l         a          1  1      a+x 

1             a 

.  Jog 

t         a  —  x 

1 

k  .  a    i  n  - 
creases 
with     in- 
crease 
of  a 

•  log           or     •  log 
t          a-x      t          a-x 

2  (Herzog) 

•log 
t          a-x 

(loc.  cit.,  p.  62) 
1-67-1-29 

log  ci  -  log  c2 

Influence    of    the 
concentration  of 
sugar,  a 

k.a  increases  with  diminution  of  a  ,  within  the 
region'  of  concentration  examined. 

As  has  been  already  stated,  B  u  c  h  n  e  r  and  M  e  i  s  e  n  - 
h  e  i  m  e  r  arrived  at  the  conclusion  that  zymase — using  the 
word  in  its  wider  sense — is  a  mixture  of  at  least  two  enzymes, 
namely,  zymase  in  a  restricted  sense  which  decomposes  fermentable 
hexoses  into  lactic  acid,  and  lactacidase,  which  breaks  down  the 
lactic  acid  into  alcohol  and  carbon  dioxide.  There  has  been 
a  considerable  amount  of  discussion  concerning  the  constituent 
chemical  processes  which  take  part  in  the  transformation  of 
sugar  into  alcohol  and  carbonic  acid.  Since  grave  objections 
have  been  advanced  to  the  intermediate  formation  of  lactic 
acid  (compare  the  critical  review  byBuchner  and  M  e  i  s  e  n  - 
h  e  i  m  e  r  in  Landw.  Jahrbiicher,  1909,  38,  Erganz.-Band  5, 
265),  nothing  final  can  be  said  as  regards  the  number  'and 
nature  of  the  participating  enzymes. 

That  a  "  co-enzyme  "  plays  an  important  part  in  fermenta- 
tion must  now  be  considered  an  established  fact.  The  same  is 
the  case  with  the  observation  that  addition  of  phosphates  to 
pressed  yeast-juice  containing  glucose  not  only  accelerates  the 
fermentation  but  also  increases  the  total  fermenting  power  or 
the  amounts  of  alcohol  and  carbon  dioxide  which  can  be  formed 
from  a  given  quantity  of  sugar  by  the  pressed  juice  (Harden 
and  Young,  Proc.  Roy.  Soc.,  1905,  77,  405;  1906,  78,  369; 
1908,  80,  299). 

These  investigators  have  studied  quantitatively  the  accelera- 
tion of  fermentation  by  phosphates.  Of  their  results,  which  are 


212 


GENERAL  CHEMISTRY   OF  THE  ENZYMES 


expressed  in  the  form  of  tables  and  curves,the  following  may  be 
cited  (Proc.  Roy.  Soc.,  1908,  80,  307): 


Time  after 
addition, 
in  minutes. 

Carbon  dioxide  evolved  in  preceding  5  minutes,  with  n  c.c.  of  0-3 
molar  potassium  phosphate  solution  added. 

n  =  0  c.c. 

n  =  10  c.c. 

n  =  15  c.c. 

5 

4-0 

11-1 

7-7 

10 

3-2 

16-0 

9-7 

15 

4-2 

20-2 

12-1 

20 

3-6 

22-4 

16-1 

25 

4-3 

17-4 

18-4 

30 

3-6 

6-6 

19-4 

35 

4-3 

4-6 

20-4 

40 

3-2 

4-7 

16-7 

45 

— 

4-5 

12-7 

50 

— 

4-2 

6-0 

55 

~ 

4-1 

4-0 

It  is  evident  that  a  marked  optimum  of  phosphate-con- 
centration exists,  the  velocity  of  fermentation  diminishing  if  this 
is  exceeded. 

The  results  of  another  series  of  experiments  are  shown  graph- 
ically in  the  curves  given  below  (Fig.  6).  The  concentrations  of 
the  solutions  were  as  follows : 


Curve  A  :  25  c.c. pressed  juice  +  5  c.c. phosphate  +15  c.c.  bicarbonate 
"      B  :  25  c.c.  +10  c.c.  +10  c.c. 

"      C  :  25  c.c.  +15  c.c.  +  5  c.c. 

"      D  :  25  c.c.  +20  c.c.  +  0  c.c. 

"  These  results  suggest  that  the  phosphate  is  capable  of  form- 
ing two  or  more  different  unstable  associations  with  the  fermenting 
complex.  One  of  these,  formed  with  low  concentrations  of  the 
phosphate,  has  the  composition  most  favourable  for  the  decom- 
position of  sugar,  whilst  the  others,  formed  with  high  concen- 
trations of  phosphate,  contain  more  of  the  latter,  probably 
associated  in  such  a  way  with  the  fermenting  complex  as  to  ren- 
der the  latter  partially  or  wholly  incapable  of  effecting  the  decom- 
position of  the  sugar  molecule." 

The  influence  of  arsenates  and  arsenites  on  the  evolution  of 
carbon  dioxide  produced  by  yeast-juice  has  also  been  studied 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       213 

in  detail  by  H  a  r  d  e  n  and  Young  (Proc.  Roy.  Soc.,  B,  1911, 
83,  451). 

"  A  striking  feature  of  the  effect  of  the  addition  of  a  phos- 
phate to  yeast-juice  is  that  the  marked  acceleration  only  con- 
tinues until  an  amount  of  carbon  dioxide  has  been  evolved  which 
is  chemically  equivalent  to  the  phosphate  added.  Moreover, 
at  the  close  of  this  period  of  enhanced  fermentation,  the  added 
phosphate  is  no  longer  present  in  a  form  precipitable  by  magnesium 
citrate  mixture,  but  has  become  converted  into  a  hexosephosphate. 


0     5     10   15   20   25   30 


50          60          70 
.Time  in  Minutes 

FIG.  6. 


Neither  of  these  phenomena  occurs  when  an  arsenate  is  substituted 
for  the  phosphate.  The  enhanced  rate  of  fermentation  continues 
long  after  an  equivalent  of  carbon  dioxide  has  been  evolved  and 
no  organic  combination  of  arsenic  is  formed. 

The  sharp  contrast  between  the  actions  of  arsenate  and  phos- 
phate is  clearly  shown,  when  the  effects  of  equivalent  amounts 
of  phosphate  and  arsenate  on  the  same  sample  of  yeast-juice 
are  directly  compared,  as  is  done  in  the  following  experiments." 


214 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


20  c.c.  Yeast-juice 


+5  c.c.  0-3  mol.  phosphate. 

+5  c.c.  0-3  mol.  arsenate. 

+0-75  c.c.  0-3  mol.  arsenate 

Time. 

Total 

Rate  per 
5  minutes. 

Total. 

Rate  per 
5  minutes. 

Total. 

Rate  per 
5  minutes. 

5 

7-1 

7-1 

11  4 

11-4 

21-7 

21-7 

10 

19-8 

12-7 

26-5 

15-1 

46 

24-3 

15 

36  1 

16-3 

43 

16-5 

71 

25 

20 

43-8 

7-7 

59 

16 

95-3 

24-3 

25 

45-7 

1-9 

75 

16-3 

119-8 

24-5 

30 

47-5 

1-8 

" 

— 

— 

— 

Formation  of  the  Phosphoric  Ester 

Contrary  to  the  opinion  of  I  wan  of  f  (H.,  1907,  50,  281; 
Centralbl.  f.  Bakt.,  1909,  II,  24,  1),  Harden  and  Young 
(Centralbl.  f.  Bakt.,  1911,  II,  26,  178)  regard  the  formation  of 
the  ester  as  due,  not  to  a  special  enzyme,  but  to  the  zymase. 
According  to  E  u  1  e  r  and  K  u  1 1  b  e  r  g  '  s  and  E  u  1  e  r  and 
O  h  1  s  e  n  '  s  experiments  with  the  extract  of  dried  yeast  (H.,  1911, 
74,  15,  1912,  76,  468),  the  enzymic  formation  of  the  phosphoric 
ester  can,  however,  be  separated  from  the  other  fermentative 
processes,  and  the  author  (H.,  1911,  74,  13)  hence  suggests 
the  name  phosphatese  for  this  synthesising  enzyme. 

If  glucose  is  fermented  by  pressed  yeast-juice  in  presence  of 
phosphates,  the  latter  very  soon  become  combined  organically. 
This  also  occurs  with  extract  of  Munich  yeast,  no  matter  whether 
this  be  dried  at  40°  or,  as  von  Lebedew  suggests,  at  25-35°; 
in  both  cases,  as  was  described  by  Harden  and  Young, 
fermentation  and  formation  of  phosphate  proceed  together. 

But  if  another  race  of  yeast  with  a  lower  fermenting  power 
is  dried  at  40°  in  a  vacuum  and  then  extracted  in  the  usual  manner, 
no  formation  of  phosphoric  ester  occurs  with  glucose  (or  mannose 
or  fructose)  (Table  a,  below) .  If,  however,  the  glucose  is  partially 
fermented  beforehand  in  absence  of  living  yeast,  organic  combina- 
tion of  the  phosphate  takes  place  (Table  6)  (E  u  1  e  r  and  O  h  1  - 
sen,  Biochem.  Z.,  1911,  36,  313). 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       215 


TABLE  a. 

25  c.c.  yeast-extract. 


20  c.c.  glucose  solution  (20%). 
10  c.c.  5%  Na2HP04. 

Minutes. 

Grms.  of  Mg2P2Or 
from  10  c.c.  of  the 
mixture. 

0 
150 
269 

0-0399 
0-0394 
0-0394 

TABLE  6. 

25  c.c.  yeast-extract. 

20  c.c.  fermenting  glucose  solution  (2Q%). 

10  c.c.  5%  Na2HP04. 


Minutes. 

Grms.  of  Mg2P2O7 
from  10  c.c.  of  the 
mixture. 

0 

0-0414 

74 

0-0134 

264 

0-0000 

Like  other  enzymes,  the  phosphatese  may  be  precipitated  from 
the  aqueous  yeast-extract  by  means  of  alcohol,  a  considerable 
proportion  of  its  activity  remaining  after  this  treatment. 

It  is  remarkable  that  the  synthetic  enzymic  action  of  the 
extract  is  greatly  increased  by  heating  it  at  40°  for  half  an  hour. 


CATALASES 

The  decomposition  of  hydrogen  peroxide  effected  by  the  action 
of  unknown  constituents — termed,  after  O  .  L  o  e  w  ,  catalases — 
of  the  animal  and  vegetable  body,  is  so  easily  followed  quanti- 
tatively, that  this  process  has  formed  the  subject  of  a  large 
number  of  investigations  and  is  to-day  one  of  the  best- 
known  enzymic  changes.  Especially  from  the  fatty  tissues  of 
animals  can  solutions  be  prepared  which,  beyond  their  ability 
to  decompose  hydrogen  peroxide,  exhibit  few  enzymic  activities; 
particularly  small  are  the  amounts  of  organic  matter  present  in 
these  liquids,  which  must  therefore  be  comparatively  pure.  Also 
blood-serum,  the  organs  of  plants,  etc.,  by  precipitation  with 
alcohol  and  suitable  treatment  of  the  precipitates,  yield  prepara- 
tions which,  per  unit  of  weight,  rapidly  decompose  hydrogen 
peroxide  (O  .  L  o  e  w  ,  Rep.  U.  S.  Dep.  of  Agric.,  1901,  No. 
68;  S  enter,  Zeitschr.  f.  physikal.  Chem.,  1903,  44,  257  and 
Proc.  Roy.  Soc.,  1904,  74,  201;  E.  J.  Lesser,  Zeitschr.  f. 
Biol.,  1906,  48,  1;  L.  van  Italie,  Soc.  Biol.,  1906,  €0,  150; 
K  a  s  1 1  e  and  Loevenhart,  Amer.  Chem.  Journ.,  1903, 
29,563;  L.von  Liebermann,  Pfliig.  Arch.,  1903,  104, 
176  and  Chem.  Ber.,  1905,  38,  1524). 

Catalase  follows  the  theoretically  simplest  formulae  and 
relations  with  an  approximation  shown  with  few  other  enzymes, 


216 


GENERAL   CHEMISTRY  OF  THE  ENZYMES 


possibly  because  the  simple  nature  of  the  chemical  process  con- 
ditions especially  simple  relations,  or  possibly  owing  to  the 
exclusion  of  any  considerable  complication  by  the  relatively 
high  degree  of  purity  of  the  enzyme.  The  most  important  general 
conclusions  arrived  at  are  as  follows : 

The  enzymic  decomposition  of  hydrogen  peroxide  in  neutral 
or  acid  solution  is  a  reaction  of  the  first  order. 

In  dilute  solutions  of  the  peroxide,  decomposition  follows  the 
law  of  mass  action  exactly.  Numerous  physico-chemical  measure- 
ments of  the  action  of  catalases  have  been  carried  out,  the  fol- 
lowing series  of  numbers  being  given  by  G.  Senter  and  by 
H.  Euler  : 


Blood-catalase.       Senter,    loc.      cit., 
p.  282.     jJs-molar  H2O2-solution. 

Fungus-catalase.       Euler,    Hofm.    Beitr., 
1905,  7,  1.      2Vmolar  H2O2-solution. 

Minutes. 

a—  x 

fc.10*. 

T=0°. 

Minutes. 

a  —  x. 

k.lW. 

r  =  i5°. 

0 

46-1 



0 

8-0 



5 

37-1 

190 

6 

6-9 

107 

10 

29-8 

192 

12 

5-8 

116 

20 

19-6 

190 

19 

5-0 

107 

30 

12-3 

193 

55 

2-5 

100 

50 

5 

194 

I  s  s  a  e  w  (H.,  1904,  42,  102)  also  obtained  very  constant 
values  of  k  with  dilute  solutions  of  hydrogen  peroxide;  with 
more  concentrated  solutions  (0-01  N  and  above)  the  constants 
diminish. 

Catalases  are  extremely  sensitive — preparations  of  different 
origins  to  varying  degrees — towards  higher  concentrations  of 
hydrogen  peroxide.  Thus,  blood-cat alase  is  appreciably  destroyed 
(perhaps  oxidised)  in  about  0-01  N-H2O2,  this  .action  being 
naturally  more  rapid  at  higher  than  at  low  temperatures,  the 
values  of  k  being  greatly  diminished  even  at  30°.  It  is  hence 
advisable  to  employ  low  temperatures  in  working  with  catalase. 
According  to  Waentig  and  Steche  (H.,  1911,  72,  226), 
this  injurious  action  is  observable  even  at  0°  in  about  N/80- 
hydrogen  peroxide  solution. 

The  reaction  constants  are,  as  they  should  be  theoretically, 
nearly  independent  of  the  concentration  of  the 
peroxide,  equal  percentage  amounts  being  decomposed  per 


EXPEKIMENTAL  DATA  OF   ENZYME  REACTIONS       217 


unit  of  time  by  equal  quantities  of  enzyme.      Senter    (loc. 
c  i  t  .  ,  p.  283)  gives  the  following  resume: 


CH20, 

/c.104 

CH2o2 

fc.103 

Cn2o2 

fc.103 

290* 

120 

iN 

175 

Io^N 

192 

1     N 

122 

AT 

188 

1 

225 

1100  N 

460 

(The  two  values  of  k  in  each  of  these  pairs  are  comparable,  one  with  the 
other,  but  not  with  those  of  the  other  pairs.) 

The  following  results,  given  by  Senter,  indicate  the 
influence  of  the  concentration  of  the  enzyme: 

Concentrations,  E,  in  the  proportions     3  6  8          24 

fc.104  28          58          72          230 

k  :  C  9-3        9-7        9-0        9-6 

Within  the  limits  of  experimental  error  the  velocities  of 
reaction  in  very  dilute  (^iir-niolar)  solutions  of  hydrogen  peroxide 
are  therefore  proportional  to  the  concentrations  of  the  enzyme. 
With  stronger  peroxide  solutions,  Senter  found  deviation  from 
this  rule.  While  fc.104  is  about  100  in  weak  catalase  solutions, 
its  value  in  presence  of  the  threefold  quantity  of  enzyme  is  approx- 
imately 360.  That,  under  these  conditions,  the  velocity  of  reac- 
tion increases  more  rapidly  than  the  concentration  of  the  enzyme, 
is  confirmed  by  experiments  made  by  Bach,  who  investigated 
the  dynamics  of  the  catalases  with  a  preparation  from  beef-fat 
(Chem.  Ber.,  1905,  38,  1878). 

With  N/210-peroxide  solutions,  I  s  s  a  e  w  observed  deviation 
from  proportionality  in  the  opposite  direction,  that  is,  a  slower  increase 
of  the  velocity  than  of  the  enzyme-concentration.  But  the  yeast-catalase 
he  employed  must,  like  all  yeast-enzymes,  have  been  very  impure. 

As  was  shown  originally  by  L  o  e  w  ,  catalases  are  extremely 
sensitive  to  acids.  The  action  of  acid  has  also  been  the  subject  of 
numerous  quantitative  investigations,  which  show  that  the  enzyme 
is  not  permanently  changed  by  acids,  neutralisation  of  these  being 
accompanied  by  return  of  the  catalytic  activity. 


218 


GENEKAL  CHEMISTRY  OF  THE  ENZYMES 


100  c.c.  of  catalase  solution  were  poisoned,  mixed  with  100  c.c. 
of  H2O2  solution  and  25  c.c.  then  titrated;  concentration  of 
H2(>2  in  the  mixture  0-005  -normal. 


snVu  norm.  HC1. 

Tffs0o  norm.  HC1. 

Without  addition. 

Minutes 

CH202 

/c.104 

Minutes 

CH202 

/c.104 

Minutes 

Cn2o2 

fc.104 

0 

27-9 



0 

27-9 



0 

27-9 



70i 

25-3 

6 

15* 

25-6 

26 

5| 

19-8 

278 

136 

23-0 

6 

35| 

22-4 

27 

15 

10-5 

275 

195 

20-9 

6 

66^ 

18-7 

26 

25i 

5-8 

258 

1305 

3-0 

6 

185 

10-1 

24 

Yuggg-norm.  HOI. 

Incubation  period,  2  hours;  then  a  small  excess 
of  NaOH  added,  and  afterwards  the  H2O2. 

Without  addition. 

Minutes. 

CH«O! 

fc.104 

Minutes. 

CH2O2 

fc.104 

0 

28-7 



0 

28-7 



5| 
15* 

23-8 
18-5 

121 
113 

5 

25-2 
16-9 

120 
121 

26i 

14-9 

86 

31* 

11-9 

127 

41* 

11-7 

70 

53 

6-5 

120 

As  is  shown  by  the  latter  table,  the  catalase  is  not  changed 
by  the  dilute  hydrochloric  acid  solution,  even  after  two  hours. 
The  incubation  period,  that  is,  the  time  during  which  the  enzyme 
remains  in  contact  with  the  acid,  has  been  repeatedly  shown 
to  be  without  influence  on  the  subsequent  activity  of  the  enzyme. 

The  catalytic  power  of  catalases  is  also  reduced  by  quite 
small  amounts  of  baryta. 

Temperature,  10°  TT£O  norm.  Ba(OH)2.  ah  norm.  Ba(OH)2.  Without 

Catalase  from  fatty  tissue  Incubation  period,     Incubation  period,    addition 

(E  u  1  e  r  ,  Hofm.  Beitr.,  15  minutes. 

1905,7,12)  fc.!03  =  40 


40  minutes. 
A;.103=40 


of  baryta. 
/c.103  =60 


This  sensitiveness  towards  alkali  varies  considerably  with  catalases 
of  different  origins. 

A  very  slight  increase  of  the  alkalinity,  however,  appears  to 
Increase  the  velocity  of  the  decomposition  to  some  extent.  Thus, 
the  author  found  (1  o  c  .  c  i  t . )  that  the  velocity  of  reaction  of 
catalase  from  Boletus  scaber  is  doubled  by  suspending 
pure  magnesium  hydroxide  in  the  solution. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       219 

As  is  brought  out  most  clearly  by'Sorensen's  results 
'(Biochem.  Z.,  1909,  21,  131),  the  optimal  activity  of  catalase 
is  always  exhibited  in  almost  neutral  solution. 

The  actions  of  acids  and  bases  on  this  enzyme  depend,  in 
all  probability,  on  the  formation  of  salts  by  these  electrolytes 
with  the  catalase. 

Bach  (Chem.  Ber.,  1905,  38,  1878)  has  also  investigated 
catalase  quantitatively,  his  results  in  general  agreeing  with  those 
required  by  theory. 

OXYDASES 

Substances  of  unknown  constitution  and  composition,  formed 
in  the  animal  and  vegetable  kingdoms  and  capable  of  initiating 
oxidation  changes,  have  been  designated  oxydases,  generally 
without  any  definite  proof  of  their  mode  of  action;  their  sensitive- 
ness to  heat  was,  however,  established  and  they  were  then  classed 
with  the  enzymes.  That  these  substances  effected  catalytic 
acceleration  of  oxidation  processes  was  seldom,  and  could  indeed 
only  with  difficulty  be,  proved,  especially  when  isolated  con- 
stituents of  an  organ  or  juice  were  not  examined.  On  the  other 
hand,  many  other  enzymes  are  not  catalysts  in  the  strictest  sense 
of  the  word,  so  that  no  limit  could  easily  be  drawn.  The  wide 
distribution  of  oxydases  in  vegetable  and  animal  organisms  ren- 
ders it  probable  that  these  substances  perform  an  important 
function  in  all  life-processes;  but  what  this  function  is,  what 
reactions  the  oxydases  bring  about  in  the  living  animal  or  plant, 
still  remains  unknown.  The  members  of  this  class  of  bodies 
which  have  as  yet  been  obtained  exhibit  a  somewhat  limited 
sphere  of  action. 

Aldehydases 

Medwedew  has  made  a  very  complete  study  of  the  oxidising 
agent  of  the  liver,  with  reference  to  its  action  on  salicylic  aldehyde 
(Pfliig.  Arch.,  1896,  65,  249;  1899,  74,  193;  1900,  81,  540  and 
1904,  103,  403). 

As  regards  the  final  state  or  equilibrium,  the  following  results 
were  obtained.  Case  1 :  relatively  high  concentration  of  salicylic 
aldehyde  in  neutral-acid  solution.  The  concentration  of  the 
oxidation  product  (salicylic  acid)  is  inversely  proportional  to  the 
square-root  of  the  amount  of  substance  to  be  oxidised  and  approx- 


220  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

imately  proportional  to  the  square  of  the  concentration  of  the 
aldehydase. 

Case  2:  relatively  high  concentration  of  salicylic  aldehyde  in 
neutral-alkaline  solution.  One  and  the  same  quantity  of  the 
oxydase  gives  at  the  end  of  the  reaction,  that  is,  on  complete 
exhaustion  of  the  oxidising  power,  one  and  the  same  amount  of 
acid,  no  matter  what  the  concentration  of  the  aldehyde. 

In  relation  to  the  velocity  it  was  found :  (a)  If  to  the  quantity 
of  oxydase  m  an  excess  of  aldehyde  a  is  added,  the  velocity  of 
oxidation  is  proportional  to  the  square-root  of  the  concentration 
of  aldehyde.  In  Medwedew's  opinion,  liver-oxydase  is 
inactivated  by  the  oxidation.  But  this  must,  in  reality,  only 
be  a  question  of  the  consumption  of  an  oxidising  agent  obtained 
from  the  liver. 

(b)  If  the  concentration  of  aldehyde  is  less  than  that  which 
the  oxydase  present  is  able  to  oxidise,  the  velocity  of  oxidation 
is  proportional  to  the  square  of  the  aldehyde-concentration,  so 
that  dx  :  dt  =  k(a—x)2,  where  x  is  the  concentration  of  the  alde- 
hyde changed  up  to  time  t  and  a  the  initial  concentration. 

How  far  these  relations  are  quantitatively  reproducible 
and  hence  are  independent  of  the  non-controllable  composition 
of  the  liver-extract,  and  how  far  this  interpretation  of  the  numbers 
obtained  is  fitting,  remain  undecided.  Doubts  have,  however, 
been  expressed  on  these  questions  (compare  Bach's  remarks, 
Chem.  Ber.,  1905,  38,  3791)  ;  but  D  o  n  y  -  H  e  n  a  u  1 1  and 
van  Duuren's  experiments  have  led  to  different  results 
(Bull.  Acad.  roy.  Belgique,  1907,  577). 

Slowtzpff's  observation  (H.,  1900,  31,  227)  that  "  potato- 
laccase  "  oxidises  paraphenylenediamine  solution  with  a  velocity 
proportional  to  the  square-root  of  the  quantity  of  this  "  laccase," 
also  appears  to  the  author  to  be  insufficiently  proved.  What 
Slowtzoff  investigated  must  have  been  a  very  impure 
mixture  of  a  peroxydase  and  a  substance  Allied  toMedicago- 
laccase.  The  author  obtained  the  latter  component  from  potatoes 
by  precipitation  with  alcohol.  The  nitrogen-content  of  the 
first  precipitate  amounted  to  2  •  84% ;  after  solution  of  the  prepa- 
ration, treatment  with  animal  charcoal  and  further  precipitation 
with  alcohol,  the  proportion  of  nitrogen  present  fell  to  1-6%, 
whilst  the  ability  to  accelerate  the  oxidation  of  hydroquinone 
solutions  in  presence  of  manganese  salts  remained  undiminished. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       221 


As  regards  the  "  laccases,"  the  investigations  of  E  u  1  e  r 
and  B  o  1  i  n  (H.,  1908,  57,  80;  1909,  61,  1  and  72)  show  that  the 
oxydase  of  Rhus  vernicifera  and  Rhus  succe- 
d  a  n  e  a  differs  considerably  from  those  of  Medicago 
s  a  t  i  v  a  ,  etc.,  which,  according  to  Bertrand,  are  also, 
termed  laccases.  As  Bertrand  himself  showed,  the  Rhus- 
preparations  are  rich  in  manganese,  whilst  this  is  not  the  case 
with  laccases  of  the  Medicago  -type.  The  former  alone 
turn  guaiaconic  acid  solutions  blue  directly  and  redden  solutions 
of  guaiacol.  This  effect  of  Rhus  -laccase  and  also  the  power 
to  transfer  molecular  oxygen  to  phenols  (hydroquinone,  pyrogallol) 
are  destroyed  by  heating  the  solution  to  100°  for  a  short  time, 
whilst  Medicago  -laccase  is  unchanged  by  heating. 

In  the  case  of  Rhus  -laccase,  Bertrand  (Bull.  Soc. 
Chim.,  1897,  [iii],  17,  619)  established  approximate  proportionality 
between  the  manganese-content  and  the  catalytic  activity. 
The  extraordinary  sensitiveness  of  this  preparation  to  acid  is 
shown  by  the  following  measurements  (Ann.  Inst.  Pasteur,  1907, 
21,  673). 

The  oxidation  of  guaiacol  to  tetraguaiacoquinone  was  measured. 
To  a  2%  guaiacol  solution  were  added  a  little  (0  •  1  grm.  per  litre)  laccase 
(from  Rhus  succedanea)  and  sufficient  sulphuric  acid  to  give 
the  mixture  the  acidity  shown  in  the  table.  The  numbers  represent 
the  intensities  of  the  red  colour — measured  in  a  colorimeter — produced 
after  5  hours  by  the  tetraguaiacoquinone  formed. 


Normality  of  the  acid. 

Serie3  1. 

Series  2. 

0 

1 

100 
100 

100 

73.4 

48-8 
20-3 

100 
90-9 

75-2 
60-4 
60-4 
60-4 

500,000 
1 
400,000 
1 

200,000 
1 

100,000 

1 

50,000 

222 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Oxydases  of  the  M  e  d  i  c  a  g  o  -type,  in  presence  of  neutral 
manganese  salts,  accelerate  the  transference  of  molecular  oxygen 
to  the  poly  phenols. 

A  representation  of  the  course  followed  by  this  action  is 
given  by  the  following  experiments : 

50  c.c.  of  a  solution,  0-2-normal  as  regards  hydroquinone  and 
0-001 -equivalent  normal  as  regards  manganese  acetate,  and 
containing  a  weighed  quantity  of  Medicago  -oxydase,  were 
shaken  in  a  glass  vessel  from  which  the  air  had  been  replaced 
by  pure  oxygen.  The  oxidation  of  the  hydroquinone  to  quinone 
or  quinhydrone,  produced  by  the  manganese  and  oxydase,  was 
measured  by  the  diminution  in  the  volume  of  oxygen  in  the  vessel. 


0-2  grm.  oxydase  per  50  c.c. 

0  •  1  grm.  oxydase  per  50  c.c. 

Minutes. 

Oxygen  absorbed 
(c.c.). 

Minutes. 

Oxygen  absorbed 
(c.c.). 

5 

1-8 

5 

1-0 

10 

2-3 

10 

1-7 

15 

3-0 

15 

2-2 

20 

4-1 

20 

2-5 

30 

5-9 

30 

3-1 

It  has  been  shown  that  Medicago  -oxydase  is  a  mixture 
of  calcium  salts  of  organic  mono-  and  poly-basic  hydroxy-acids, 
among  which  are  glycollic,  citric,  malic  and  mesoxalic  acids. 
The  catalytic  action  of  these  pure  salts  corresponds  closely  with 
that  of  Medicago  -oxydase,  as  is  shown  by  the  following 
numbers  obtained  under  the  conditions  described  above. 


0-2  grm.  calcium  oxalate  per  50  c.c. 


0  •  1  grm.  calcium  glycollate      ] 

0-05  grm.  calcium  malate          ^  per  50  c.c. 

0-05  grm.  calcium  mesoxalate  J 


Minutes. 

Oxygen  absorbed 
(c.c.). 

Minutes. 

Oxygen  absorbed 
(c.c.) 

5 

1-8 

5 

2-3 

10 

2-9 

10 

3-4 

20 

4-5 

15 

4-1 

30 

5-7 

20 

4-8 

30 

5-9 

As  is  well  known,  the  oxidation  of   polyphenols,  either  alone 
or,  more  markedly,  in  presence  of  manganese  salts,  is  considerably 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       223 

increased  by  small  quantities  of  alkali,  so  that  especial  care  must 
be  taken  as  regards  the  neutrality  of  the  oxydase  and  of  the  calcium 
salts.  In  the  above  investigation  they  were  absolutely  neutral. 

Since  the  salts  of  all  the  aliphatic  hydroxy-acids  appear  to  be 
more  or  less  active  in  this  direction,  oxydases  of  this  group  must 
be  of  very  frequent  occurrence  in  the  vegetable  kingdom. 

The  indophenol  reaction,  which  was  first  employed  by 
Ehrlich  in  1885  and  consists  in  the  formation  of  indophenol 
from  a-naphthol  and  ^-phenylenediamine,  has  been  used  recently 
by  V  e  r  n  o  n  (Journ.  of  PhysioL,  1911,  42,  402)  as  the  basis  of 
a  quantitative  method.  With  the  help  of  his  method,  this  author 
has  investigated  quantitatively  numerous  oxidation  phenomena 
induced  by  tissues. 

According  to  the  concentration  of  the  substrate  (naphthol 
and  diamine),  the  amount  of  indophenol  formed  was  found  to  be 
proportional  either  to  the  square  of  the  quantity  of  enzyme,  or  to 
this  quantity  itself  or  to  its  square  root. 

PEROXYDASES 

Presumably  still  more  widespread  and  consequently  of  more 
general  action  are  those  substances  of  the  animal  and  vegetable 
body  which  activate  'peroxides,  including  hydrogen  peroxide, 
i.e.,  transfer  the  peroxide-oxygen  to  other  substances;  these  are 
termed  peroxydases. 

The  changes  which  the  peroxydases  themselves  undergo, 
during  this  transference  of  oxygen  from  the  peroxides  to  sub- 
stances like  a-guaiaconic  acid,  are  unknown,  and  it  has  often  been 
doubted  whether  the  peroxydases  should  really  be  regarded  as 
catalysts  and  as  enzymes.  Our  conception  of  the  latter,  in 
particular,  is  so  indefinite,  that  at  the  present  time,  when  so  little 
is  known  concerning  the  exact  chemical  nature  of  the  peroxydases, 
discussion  of  this  question  is  of  little  value. 

Owing  more  especially  to  the  work  of  J.  Wolff  and  E.  d  e 
S  t  o  e  c  k  1  i  n  (C.  R.,  1911,  153,  139),  substances  such  as  potas- 
sium ferrithiocyanate,  K3Fe(CNS)6,  are  however  known  which 
are  not  of  organic  origin  and  yet  behave  like  the  peroxydases.1 

1  It  appears  to  be  established  that,  in  most  biological  oxidations  which  are 
regarded  as  enzymic,  the  true  oxidising  agent  is  peroxydic  in  character. 
This  view  was  advanced  almost  simultaneously  by  C  h  o  d  a  t  and  Bach 


224  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

We  shall  proceed  at  once  to  a  consideration  of  the  numerical 
relations — due  to  C  h  o  d  a  t  and  Bach  and  their  pupils — 
characterising  the  action  of  these  enzymes. 

In  the  experiments  which  will  first  be  described  (Chem. 
Ber.,  1904,  37,  1342),  definite  quantities  of  peroxydase  (from 
horseradish),  hydrogen  peroxide  and  pyrogallol  were  mixed  in 
aqueous  solution, .  the  purpurogallin  formed  in  24  hours  being 
collected  on  a  tared  filter,  washed  with  100  c.c.  of  water,  and 
dried  at  110°  until  of  constant  weight.  Pyrogallol  is  not  appre- 
ciably attacked  by  either  peroxydase  or  hydrogen  peroxide  alone. 

Three  series  of  experiments  were  carried  out : 

A.  With   variation  only  of  the   amount   of  peroxydase. 

B.  With  variation  only  of  the  amount  of  hydrogen  peroxide. 

C.  With  variation  only  of  the  amount  of  pyrogallol. 
Temperature,  15-17°.     Volume  of  the  mixture,  35  c.c. 

A 

HYDROGEN  PEROXIDE,  0-10  GRM.;  PYROGALLOL,  1  GRM. 

Amount  of  peroxydase,  in  grm. : 

0-01       0-02       0-03       0-04       0-05       0-06       0-07       0-08       0-09       0-10 
Purpurogallin  formed,  in  grm.: 

0-021     0-042     0-066     0-083     0-102     0-123     0-145     0-166     0-167     0-162 

B 

PEROXYDASE,  0-10  GRM.;  PYROGALLOL,  1  GRM. 

Hvdrogen  peroxide,  in  grm.: 

0-01       0-02      0-03       0-04       0-05       0-06       0-07       0-08       0-09       0-10 
Purpurogallin,  in  grm.: 

0-205     0-42       0-60       0-78       0-99       0-121     0-142     0-168     0-168     0-165 

Experiments  A  show  very  clearly  that  with  a  constant  amount 
(in  excess)  of  hydrogen  peroxide,  the  yields  of  purpurogallin  are 
exactly  proportional  to  the  quantities  of  peroxydase  employed; 
whilst,  with  varying  amounts  of  hydrogen  peroxide  and  a  constant 
quantity  (in  excess)  of  peroxydase,  the  amount  of  change  is,  as 
experiments  B  show,  proportional  to  the  former. 

An  excess  of  peroxydase  or  of  hydrogen  peroxide  is  without 
influence  on  the  oxidising  capacity  of  the  system  peroxydase- 
hydrogen  peroxide.  From  these  results  Bach  and  C  h  o  d  a  t 

(Chem.  Ber.,  1902,  35,  1275,  2487,  3943;  1903,  36,  600,  606,  1756)  and  by 
K  as  tie  and  Loevenhart  (Amer.  Chem.  Journ.,  1901,  26,  539). 
The  application  of  these  noteworthy  theories  is  often  hindered  by  the  un- 
certainty of  their  chemical  foundations,  so  that  the  theoretical  results  need 
not  be  repeated  here. 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       225 

drew  the  conclusion  that  peroxydase  and  hydrogen  peroxide  take 
part  in  the  reaction  always  in  constant  relations.  They  arrived 
thus  at  the  hypothesis  which  had  been  previously  advanced 
by  K  a  s  1 1  e  and  Loevenhart  (Amer.  Chem.  Journ.,  1901, 
26,  593),  namely,  that  the  peroxydase  forms  with  the  hydrogen 
peroxide  a  definite  compound  exhibiting  more  energetic  oxidising 
properties  than  the  peroxide  alone.  The  result  obtained  by  the 
two  first-named  investigators — that  excess  of  hydrogen  peroxide 
does  not  affect  the  change — indicates  that  the  peroxydase 
is  not  used  up  during  the  course  of  the 
oxidation. 

c 

PEROXYDASE,  0-10  GRM.;  HYDROGEN  PEROXIDE,  0-10  GRM. 

Pyrogallol,  in  grm.  1-5  2  3  4 

Purpurogallin,  in  grm.  0-205  0-203  0-208  0-202 

From  the  results  of  experiments  C  it  is  seen  that  the  concentra- 
tion of  the  pyrogallol  is  without  influence  on  the  magnitude  of 
the  change. 

Since  the  formation  of  purpurogallin  and  the  procedure 
described  above  are  not  suitable  for  the  determinination  of  the 
time-law  of  peroxydase-action,  Bach  and  C  h  o  d  a  t  (Chem. 
Ber.,  1904,  37,  2434)  chose  for  this  purpose  the  oxidation  of 
hydrogen  iodide. 

Five  c.c.  of  a  solution,  g^^-normal  with  respect  to  acetic 
acid  and  hydrogen  peroxide,  were  added  to  45  c.c.  of  a  solution 
containing  ^^-equivalent  of  potassium  iodide  and  varying 
quantities  of  peroxydase.  The  iodine  separated  after  a  certain 
time  was  estimated  by  titration  with  thiosulphate. 

1-25 

-EQUIVALENT  PEROXYDASE  * 
luU.uUU 

Minutes 1  2  4  6  8  10  12          20 

c.c.  thiosulphate.   1-3        2-4        4-4        6-1         7-1         7-9        8-1         8-4 

PARALLEL  EXPERIMENT  WITHOUT  PEROXYDASE 

Minutes 2  4  6  8  10  12          20 

c.c.  thiosulphate 0-25        0-45      0-6        0-8        0-9         1-1         1-6 

2-5.10~5-EQUIVALENT  PEROXYDASE 

Minutes 1  2  4  6  8  10  20 

c.c.  thiosulphate 2-4        4-7        8-4       10-2       11-7       11-8       12-3 

*  The  peroxydase-preparation  was  the  same  as  irf  the  above  experiments.  It  activated 
exactly  its  own  weight  of  hydrogen  peroxide,  or,  according  to  Bach  and  C  h  o  d  a  t  '  s 
mode  of  expression,  it  had  the  activating  power  1. 


226  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

5 . 10~~5-EQUIVALENT  PEROXYDASE 

Minutes '. . .     1  2  46  8          10          20 

c.c.  thiosulphate 4-9        8-9      14-0      14-7      15-0      13-1       15-4 

10.10~5-EQUIVALENT  PEROXYDASE 

Minutes 1  2  4  6  8          10          20 

c.c.  thiosulphate 10-1       15-7      17-0      17  1       17-2      17-2      17-4 

15.10~5-EQUIVALENT  PEROXYDASE 

Minutes 1246  8          10          20 

c.c.  thiosulphate 15-0      18-6      19-1       19-1       19-2      19-2      19-4 

It  will  be  seen  first  of  all  that,  in  the  oxidation  of  hydriodic 
acid  just  as  in  that  of  pyrogallol,  the  peroxydase  ultimately  loses 
its  activity,  since  after  some  time  the  procedure  of  the  reaction 
becomes  exactly  that  exhibited  when  no  peroxydase  is  added; 
the  rate  at  which  the  activity  of  the  peroxydase  diminishes 
increases  with  the  concentration  of  the  peroxide.  Hence  the 
results  obtained  with  different  concentrations  of  peroxydase 
are  comparable  only  for  those  phases  of  the  reaction  where  the 
enzyme  still  exerts  almost  its  full  activity 

This  is  still  the  case  at  the  end  of  the  first  minute,  after  which 
time    the     magnitudes    of     the     change    are    exactly— 
within     the    limits    of    experimental    error- 
proportional    to     the    amounts  of  peroxydase: 

Peroxydase-concentration  X 105 2-5        5  10  15 

Amount  of  change  after  1  minute  (c.c.) 2-4        4-9  10-1         15-0 

Comparison  of  the  final  states  reached  in  the  system 
hydriodic  acid-peroxydase-hydrogen  peroxide  shows  that  the 
amount  of  the  product  of  the  reaction  (iodine)  is  not,  as  with  the 
oxidation  of  pyrogallol,  directly  proportional  to  the  quantity 
of  peroxydase,  but  increases  more  slowly  than  the  latter.  It 

/y» 

is  to  be  noted  that  the  activating  power  of  the  peroxydase,  - 

a 

(where  x  denotes  the  amount  of  hydrogen  peroxide 
activated  by  a  weight  a  of  peroxydase),  is  considerably  greater 
in  the  oxidation  of  hydriodic  acid  by  hydrogen  peroxide  than  in 
the  oxidation  of  pyrogallol. 

These  experiments  with  hydriodic  acid  were  extended  by 
Bach  (Chem.  Ber.,  1904,  37,  3785),  who  determined  the 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       227 


separation  of  iodine  occurring  in  10  minutes  at  22°  in  differently 
concentrated  mixtures  of  HI,  H202  and  horseradish-peroxydase ; 
his  results  are  collected  in  the  following  table : 

Increase  of  the  change  measured  in  c.c.  of  0-01  N-thiosulphate. 


Peroxy- 
dase. 

I. 

12-5HI. 

II. 

25HI. 

ill. 

37-5HI. 

IV. 
50HI. 

V. 
75  HI. 

VI. 
100HI. 

A 

1-25 

3-1 

4-4 

5-0 

5-4 

5-3 

5-4 

B 

2-5 

5-1 

7-1 

7-9 

8-5 

8-6 

8-5 

C 

5-0 

7-0 

9-5 

11-3 

13-2 

13-6 

13-6 

D 

10-0 

9-3 

12-9 

15-7 

17*9 

21-4 

25-1 

E 

15-0 

10-9 

15-9 

18-5 

21-6 

26-0 

29-3 

F 

21-0 

10-7 

17-6 

21-4 

25-5 

29-1 

33-6 

G 

25-0 

10-8 

17-8 

24-0 

28-4 

32-6 

37-1 

Control 

— 

0-7 

1-4 

2-0 

2-7 

4-1 

5-4 

The  increase  in  the  amount  of  action  in  a  given  time  rises 
both  with  the  concentration  of  the  peroxydase  and  with  that  of  the 
hydriodic  acid  and,  for  each  concentration  of  enzyme  and  acid, 
attains  a  certain  limiting  value  and  then  remains  constant;  it 
must  therefore  be  concluded  that  peroxydase,  hydriodic  acid 
and  hydrogen  peroxide  react  together  in  definite  proportions. 

It  is  readily  seen  from  the  preceding  table  that  the  pro- 
duct of  the  concentrations  is  (within  certain 
limits)  constant;  for  instance,  DXl  =  CXlI,  etc. 

Finally,  comparison  of  the  concentrations  of  hydriodic  acid 
which  correspond  with  different  increments  in  the  change  shows 
that  these  increments  are  almost  exactly  proportional  to  the 
square-roots  of  the  concentrations  of  the  acid;  this  is  evident 
from  the  following  summary: 


I. 

II. 

III. 

IV. 

V. 

VI. 

Series  D  

9-2 

12-9 

15-7 

17-9 

21-4 

25-1 

Calculated  

9-2 

13-0 

15-9 

18-6 

22-4 

25-9 

Series  E  

10-9 

15-9 

18-5 

21-6 

26-1 

29  3 

Calculated  

10-9 

15-3 

18-8 

21-8 

26-5 

30-7 

Bach  did  not,  however,  obtain  such  regularity  with  another 
preparation,  this  giving  a  different  relation,  which  had  not  pre- 
viously been  observed. 


228 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Increase  of  the  change  measured  in  c.c.  0-01  N-thiosulphate. 


Peroxy- 
dase. 

12-5HI. 

II. 

25HI. 

ill. 

37-5HI. 

IV. 
50HI. 

v. 

75HI. 

VI. 

loom. 

A 

1-25 

1-2 

2-4 

3-3 

4-2 

4-2 

4-1 

B 

2-50 

2-2 

4-2 

6-1 

8-1 

8-3 

8-2 

C 

5-0 

3-6 

6-0 

9-4 

.12-1 

15-2 

15-8 

D 

10-0 

4-4 

8-3 

12-2 

14-6 

20-7 

25-9 

E 

15-0 

5-0 

9-6 

13-8 

18-5 

27-4 

36-6 

F 

20-0 

5-0 

10-1 

15-1 

20-1 

30-2' 

41-0 

G 

25-0 

5-1 

10-2 

15-6 

20-4 

30-0 

40-8 

Control 

— 

0-7 

1-6 

2-2 

2-9 

4-1 

5-6 

The  results  of  series  F  and  G  show  that,  after  the  p  e  r  - 
oxydase-maximum  is  reached,  the  increase 
in  the  amount  of  t  r  a  n  s  f  o  r  m  a  t  i  o  n  i  s  e  x  a  c  1 1  y 
proportional  to  the  concentration  of  the 
hydriodic  acid. 

For  the  complete  utilisation  of  peroxydase,  a  definite  acidity 
(concentration  of  the  hydrogen-ions)  of  the  liquid  is  necessary, 
the  nature  of  the  anion  of  the  acid  being  without  influence. 

The  darkening  of  many  plant-juices  owing  to  enzymic  oxida- 
tion was  found  by  G  r  a  f  e  and  Weevers  to  be  conditioned 
by  the  presence  of  catechol  and  this  discovery  has  recently  been 
confirmed  by  Miss  Wh  el  dale  (Proc.  Roy.  Soc.,  B,  1911,  84, 
122).  The  study  of  the  oxidation  of  catechol  by  plant-juices  from 
a  kinetic  standpoint  would  be  of  great  interest. 

TYROSINASE 

In  conjunction  with  an  investigation  on  melanotic  pigments 
and  the  enzyme  formation  of  melanins,1  O.  von  Ftirth  and 
E.  Jerusalem  (Hofm.  Beitr.,  1907,  10,  131)  studied  the 
mode  of  action  of  tyrosinase.  The  very  complicated  relations 
found  by  these  authors  led  Bach  (Chem.  Ber.,  1908,  41,  216, 
221),  who  regarded  tyrosinase  as  a  mixture  of  an  oxygenase  and 
a  peroxydase,  to  establish  the  conditions  of  action  of  this  enzyme. 

The  amounts  of  melanin  formed  were  determined  as  follows:  The 
juice  was  diluted  tenfold  with  distilled  water  and  10  c.c.  of  this  solution 

1  A  step  towards  the  elucidation  of  the  chemistry  of  tyrosinase  action 
has  been  made  in  a  valuable  investigation  by  Abderhalden  and 
Guggenheim  (H.,  1907,  54,  331). 


EXPERIMENTAL  DATA  OF  ENZYME  REACTIONS       229 


mixed  with  10  c.c.  of  tyrosine  solution  (containing  0-05%  of  tyrosine 
and  0-04%  of  sodium  carbonate)  and  30  c.c.  of  water;  after  24  hours, 
the  solution  was  acidified  with  1  c.c.  of  10%  sulphuric  acid  and  titrated 
with  0-002-normal  permanganate  until  the  latter  was  decolorised. 

The  tyrosinase  was  extracted  from  Russula    delica. 

1.  Dependence  of  melanin-formation  on  the  concentration 
of  tyrosinase. 

Into  each  of  a  series  of  eight  beakers  were  placed  10  c.c.  of  the 
tyrosine  solution  and  a  certain  quantity  of  the  enzyme  solution, 
the  volume  being  then  made  up  to  50  c.c.  with  water. 

Permanganate  solution  used,  in  c.c. 


I. 

II. 

.III. 

IV. 

V. 

VI. 

VII. 

VIII. 

Volume  of  enzyme  solution 
(c.c.)  

0-5 

1-0 

1-5 

2-0 

5-0 

10-0 

15-0 

20-0 

A    After  24  hours 

10-8 

14-2 

17-3 

19-8 

25-8 

30-4 

33-6 

35-8 

B.  After  48  hours  

13-2 

16-0 

17-8 

20-4 

25-6 

31-2 

34-4 

35-4 

These  results  show  that  :  (1)  the  amount  of  the  product  of 
the  reaction  increases  with  that  of  the  enzyme,  although  more 
slowly  than  the  latter,  and  (2)  the  rapidity  with  which  the  reaction 
comes  to  a  standstill  increases  with  the  concentration  of  the 
enzyme.  In  these  respects,  tyrosinase  behaves  similarly  to  horse- 
radish-peroxydase  (see  p.  227). 

2.  Velocity  of  reaction  and  concentration  of  enzyme:  Three 
750  c.c.  Erlenmeyer  flasks  were  each  charged  with  100  c.c.  of 
tyrosine  solution,  a  certain  volume  of  enzyme  solution,  and  water 
up  to  a  volume  of  500  c.c. 


Quantity 
Time  in  1 

of  enzyme  solution  per  500  c.c.  . 
lours  :     1 

10  c.c. 

c.c. 

0-0 

20  c.c. 
c.c. 
1-4 

30  c.c. 

c.c. 

2-8 

2  

0-0 

3-9 

5-7 

3 

1-6 

5-8 

8-8 

4  

2-7 

7-8 

11-1 

6  

5-5 

11-1 

16-1 

*  ' 

9  

9-4 

16-3 

20-8 

14  

15-9 

19-0 

22-3 

24  

16-0 

19-9 

°2-8 

Volume  of  permanganate  solution 
required. 


230 


GENERAL  CHEMISTRY  OF   THE  ENZYMES 


A  reaction  constant  can  scarcely  be  calculated  with  any 
degree  of  certainty,  since  the  position  of  the  end-value  is  doubtful. 
But  if  the  times  corresponding  with  equal  amounts  of  change  are 
compared,  inverse  proportionality  between  the  amount  of  enzyme 
and  the  time  of  reaction  is  clearly  evident:  the  product  of  the 
quantity  of  enzyme  and  the  time  is  hence  constant.  That  the 
initial  and  final  stages  of  the  reaction  must  be  disregarded  is 
explained  by  the  slower  initiation  of  the  reaction  with  low  than 
with  higher  concentrations  of  enzyme,  whilst  these  higher  con- 
centrations lead  to  more  rapid  inactivation  of  the  enzyme. 
3.  Velocity  of  reaction  and  concentration  of  substrate: 
Three  flasks,  containing  25,  50  or  75  c.c.  of  the  tyrosine  and 
30  c.c.  of  enzyme  solution  diluted  to  500  c.c. 


Volume  of  permanganate 
solution  required. 

Volume  of  tyrosine  solution,  in  c.c  
After    1  hour 

25 
1-0 
3-1 

4-8 
7-0 

8-4 

50 
1-9 

6-3 
9-4 
12-4 
12-6 

75 
3-0 
9-2 
13-9 
14-7 
16-2 

1  '       3  hours                

"       5     "     

"       8     " 

11     24     "     

With  a  constant  concentration  of  enzyme,  the  quantity  of 
melanin  formed  per  unit  of  time  is — apart  from  the  final  stages 
of  the  reaction — proportional  to  the  amount  of  tyrosine  present. 

Tyrosinase  hence  corresponds  well  with  the  law  of  mass  action. 


OXIDATION   OF  XANTHINE 


In  conclusion,  it  may  be  pointed  out  that  B  u  r  i  a  n  (H., 
1905,  43,  497)  has  investigated  dynamically  the  oxidation  of 
xanthine  to  uric  acid.  This  reaction  is,  however,  not  one  of  pure 
oxidation,  so  that  mention  of  this  paper  must  suffice. 


CHAPTER  V 

INFLUENCE    OF    TEMPERATURE   AND    RADIATION    ON 
ENZYMIC    REACTIONS 

TEMPERATURE  influences  chemical  systems  in  two  ways: 
It  is,  first  of  all,  one  of  the  factors  which  determine  the  posi- 
tion of  equilibrium  between  the  substances  taking  part  in  a  rever- 
sible reaction.  The  degree  to  which  the  equilibrium  changes 
with  the  temperature  in  any  case  is  closely  related  to  the  heat- 
change  of  the  reaction.  If  the  equilibrium  constant  of  a  reaction 
is  indicated,  as  before,  by  K,  while  U  denotes  the  total  heat- 
change  determined  calorimetrically,  T  the  absolute  temperature 
and  72  the  gas-constant,  van't  Hoff's  fundamental  thermo- 
chemical  law  states  that  : 

U 


dT  '       R.T2' 

If,  therefore,  the  heat-change  accompanying  any  reaction  is 
small  —  and  this  is  the  case  with  most  enzymic  processes  —  t  h  e 
equilibrium  is  only  slightly  dependent  on 
the  temperature. 

A  much  greater  alteration  with  temperature  is  shown  by 
the  velocity  with  which  a  system  proceeds  towards  its 
equilibrium  or  final  position.  In  most  cases,  a  rise  of  temperature 
of  10°  doubles  or  trebles  this  velocity  —  a  phenomenon  to  which 
van't  Hoff  first  directed  attention.  With  non-enzymic  reac- 
tions, indeed,  still  higher  temperature-coefficients  are  observed. 
From  the  abundant  experimental  data,  the  following  figures, 
referring  to  reactions  of  biological  interest,  may  be  quoted: 

Author.  Reaction.  Jemp^      *?±10        ^ 

Price,  Svenska  Vet.  Akad.  Forh,  1899  .  .  Ethyl  acetate  +H2O  28-50°  2  •  4  17,390 
E  u  1  e  r  ,  Chem.  Ber.,  1905,  38,  2551  .....  Formaldehyde  +NaOH  50-85°  3  •  6  24,900 
S  p  o  h  r  ,  Z.  physikal.  Chem.,  1888,  2,  194  .  Inversion  of  cane-sugar  25-55°  3-6  25,600 

231 


232  GENERAL  CHEMISTRY   OF  THE  ENZYMES 

k 
The  values  of  the  quotient,     ^10,  hold   only  for  a  certain 

hi 

interval  of  temperature,  the  increase  of  the  velocity  of  reaction 
per  degree  diminishing  with  rise  of  temperature.  On  the  other 
hand,  the  constant  [L  retains  its  value  over  a  very  wide  range  of 
temperature.  This  constant  is  given  by  the  formula  derived 
theoretically  byArrhenius  and  found  to  be  generally  valid : 

/*  (T,-TA 

k2  =  kleR\  TlTz  )t (23); 

it  represents  therefore  an  exact  expression  for  the  dependence 
of  the  Velocity  of  reaction  on  the  temperature.  In  this  equation, 
ki  and  £2  denote  the  reaction-constants  at  the  absolute  tem- 
peratures TI  and  T2,  while  R  is  the  constant  of  the  gas-laws  and 
e  the  base  of  the  natural  system  of  logarithms. 

An  influence  as  great  as  on  the  velocity  of  chemical  reactions 
is  exerted  by  the  temperature  on  the  vapour  pressure  of  liquids 
and  on  the  equilibria  of  certain  dissociations.  In  the  latter 
case  this  is  explainable  on  the  assumption  that  organic  reactions 
are  also  effected  by  ions.  The  constant  [L  gives  the  heat  of 
transformation  accompanying  the  conversion  of  the  participating 
molecules  from  the  "  normal  "  into  the  "  active "  state,  and 
hence  corresponds  with  the  sum  of  the  heats  of  dissociation  of  the 
substances  taking  part.  If,  for  instance,  the  inversion  of  cane- 
sugar  is  considered,  [i  is  the  sum  of  the  heat  of  dissociation  of 
water,  t/idiss.  and  that  t/2diss.>  °f  cane-sugar  (the  latter  taken  as 
a  base).  The  heat  of  dissociation  of  water  is  13,450  Cals.;  the 
value  of  E/2diss.  is  still  unknown,  but  it  is  known  that  with  such 
extremely  weak  electrolytes  as  cane-sugar  must  be,  the  heats  of 
dissociation  are,  in  general,  approximately  of  the  same  magnitude 
as  that  of  water.  On  this  assumption  for  t/2diss.>  ^  would  be 
26,900,  while  calculation  of  the  experimental  results  according 
to  A  r  r  h  e  n  i  u  s  's  formula  leads  to  the  value  25,600  (E  u  1  e  r  , 
Zeitschr.  f.  physikal.  Chem.,  1904,  47,  353).  The  influence  of 
temperature  on  the  compound,  substrate-catalyst  (i.e.,  the 
hydrochloride,  sulphate,  etc.,  of  the  cane-sugar)  is  here  apparently 
neglected.  But,  since  the  dissociation  of  strong  acids  and  of 
salts  changes  comparatively  slightly  with  the  temperature, 
this  influence  is  determined  principally  by  the  heats  of  dissociation 
of  the  cane-sugar  and  the  water. 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION      233 

On  the  velocity  of  enzyme  reactions,  temperature  has  a 
twofold  influence  :  that  just  referred  to  and,  in  addition, 
an  action  on  the  activity  of  the  catalysing  enzyme,  which  becomes 
more  rapidly  destroyed  or  permanently  inactivated  as  the  tem- 
perature rises. 

The  processes  resulting  from  these  two  actions  were  first 
treated,  theoretically  and  experimentally,  by  Tammann 
(Zeitschr.  f.  physikal.  Chem.,  1895,  18,  426). 

The  simplest  assumption  is  that  the  enzyme  is  inactivated 
in  aqueous  solution  by  a  unimolecular  reaction,  independently 
of  whatever  else  is  in  the  solution.  So  that,  if  E  is  the  initial 
concentration  of  the  enzyme  and  y  the  concentration  at  time  t, 
then: 


hence 

E-y  =  E.e~kE,      .     .    ,     ....     (24) 

where  e  is  the  base  of  the  natural  system  of  logarithms,  and 


If  further,  a  denotes  as  usual  the  original  concentration  of 
the  substrate,  which  undergoes  decomposition  according  to 
an  equation  of  the  first  order,  it  follows  that 

v  =  ~  =  k(a-x)(E-y),     ......     (26) 

that  is,  the  velocity  with  which  the  reaction  proceeds  at  time  t 
must  be  equal  to  the  product  of  the  amounts  of  enzyme  and  sub- 
strate still  present. 

Substitution  in  the  last  equation  of  the  value  of  E  —  y  from 
Eq.  (24)  gives,  as  Tammann  showed,  the  integral  equation: 


(27) 


234 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


From  Eq.  (27),  T  a  m  m  a  n  n  calculated  the  "false  equi- 
librium," that  is,  the  final  state  of  the  enzymic  reaction,  and 
compared  the  result  with  that  determined  experimentally  with  the 
system  emulsin-salicin.  The  following  tables  are  taken  from 
those  given  by  Tammann. 

To  solutions  of  salicin,  previously  heated,  were  added  varying  quan- 
tities of  emulsin,  100  c.c.  of  each  of  the  mixtures  containing  3 -007  grms. 
of  salicin  and  the  amount  of  enzyme  shown  in  the  table. 

PERCENTAGE  OF  SALICIN  HYDROLYSED 

Emulsin  (grin.).  72  hours.  104  hours.  148  hours. 

0-250  63-4  65-1  65-4   } 

0-125  48-3  50-2  50-4    \  Temp.,  65° 

0-0312  16-4  17-0  16-8  J 


Emulsin  (grm.). 

0-250 
0-125 
0-0156 


45  hours. 

(101-2) 
97-5 
59-3 


86  hours. 

99-2 
97-5 
65-7 


166  hours. 


(100-2) 

67-6 


Temp.,  45 c 


Emulsin  (grm.).  45  hours.  93  hours.  334  hours. 

0-250                    96-5                98-0  100.2    ) 

0-125                    96-5                97-5  99-6    [  Temp.,  26° 

0-0156                  85-8                92-1  98-0  J 


As  will  be  seen,  the  higher  the  temperature,  the  sooner  the 
reaction  comes  to  a  standstill,  i.e.,  the  more  rapidly  the  enzyme 
is  inactivated. 

It  must  particularly  be  pointed  out  that  a  distinction  is  to 
be  made  between  the  permanent  non-reversible  inac- 
tivation  which  every  enzyme  undergoes,  especially  at  higher  tem- 
peratures, and  the  inactivation  due  to  the  products  of  reaction 
and  disappearing  when  these  are  removed. 
In  the  latter  case,  where  the  catalysing  enzyme  is  held  by  the 
reaction-products,  the  reaction  retards  itself  by  consuming  its 
own  catalyst.  Such  catalytic  retardation  has  been  treated  by 
O  s  t  w  a  1  d  (Lehrbuch,  II,  2,  271). 

The  differential  equation  for  this  case, 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION       235 
gives,  on  integration, 


ki(Ak2  —  k 

As  O  s  t  w  a  1  d  pointed  out,  k2x  at  the  beginning  of  the 
reaction  must  not  be  greater  than  fci,  as  otherwise  the  reaction 
does  not  take  place.  The  reaction  therefore  leads  here  to  a 
"  false  equilibrium  "  or  end-state. 

The  influence  of  rise  of  temperature  on  an  enzymic  reaction 
is  hence  two-fold:  1,  Acceleration  of  the  reaction  and,  2, 
inactivation  of  the  enzyme.  So  that  both  k  and  ks  increase 
as  the  temperature  rises,  and  when  the  influence 
of  temperature  on  an  enzymic  reaction  is 
to  be  defined,  the  temperature-coefficient 
should  be  give  n  —  a  s  was  done  by  Tammann 
—  b  oth  for  k  and  for  k  E-  The  temperature-function  of 
the  two  magnitudes  is  best  expressed  by  Arrhenius's 
formula. 

Owing  to  the  dependence  of  the  stability  of  the  catalyst  on 
the  temperature,  the  temperature-curves  of  enzymic  reactions 
differ  in  appearance  from  those  of  most  other  chemical  processes. 
Thus,  they  show  an  optimum,  the  temperature-coefficient  at  a 
certain  temperature  being  zero,  owing  to  the  increased  velocity 
of  decomposition  of  the  substrate  being  exactly  compensated 
by  the  increased  rate  of  destruction  of  the  enzyme;  further  rise 
of  temperature  is  then  accompanied  by  decrease  of  the  velocity 
of  reaction. 

It  was  assumed  on  p.  233  that  the  decomposition 
of  enzymes  obeys  the  formula  for  unimolecular  reactions.1 
This  is  best  ascertained  by  keeping  the  enzyme  for  a  certain  time 
in  aqueous  solution  at  the  temperature  to  be  investigated,  then 
mixing  it  —  where  possible  at  a  lower  temperature  —  with  the 
substrate  and  calculating  the  constant  kE  from  the  initial  velocity. 
The  values  of  kE  obtained  at  different  temperatures  then  give 
the  constant  y.  of  the  temperature-formula  of  Arrhenius 
given  on  p.  232, 


1  Whether  all  enzymes  decompose  according  to  this  law  is  still  question- 
able.    According  to  S  e  n  t  e  r  it  is  not  the  case  with  catalase. 


236  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Spontaneous  decomposition  of:          ji 

Emulsin  in  0-5%  solution 45,000    T  a  m  m  a  n  n 

Rennet  in  2%  solution  l 90,000  ] 

Trypsin  in  2%  solution 62,034  V  M  a  d  s  e  n  and  W  a  1  b  u  m  * 

Pepsin  in  2%  solution  2 75,600  J 

Yeast-invertase 72,000     E  u  1  e  r  and    a  f   U  g  g  1  a  s 

Dry  emulsin 26,300     T  a  m  m  a  n  n 

For  yeast-invertase,  E  u  1  e  r  and  K  u  1 1  b  e  r  g  (H.,  1911, 
71,  134)  have  shown  that  (JL  is  independent  of  the  impurities 
arising  from  the  yeast  and  hence  represents  a  well-defined  constant. 

Mention  must  also  be  made  of  the  measurements  made  by 
N  i  c  1  o  u  x  with  lipase  (Soc.  Biol.,  1904,  56,  701,  839,  868),  the 
value  obtained  for  [L  being  about  26,000. 

S  e  n  t  e  r  (Zeitschr.  f.  physikal.  Chem.,  1903,  44,  257)  states 
that  the  destruction  of  blood-catalase  at  55°  is  about  6-7  times 
as  rapid  as  at  45°;  [L  is  about  50,000. 

Special  emphasis  must  be  laid  on  the  high  values  of  \L  com- 
pared with  those  of  the  corresponding  constants  for 'other  reac- 
tions, such  as  those  given  on  p.  231.  The  result  obtained  with 
dry  emulsin  shows  the  slight  sensitiveness  to  heat  of  dry  enzymes 
relatively  to  that  shown  in  the  dissolved  state.  Similar  observa- 
tions of  a  qualitative  character  have  often  been  made  with  other 
enzymes.  The  numerical  values  obtained  under  these  conditions 
for  [L  are,  as  is  easily  understood,  dependent  in  a  high  degree  on 

1  Merely  by  shaking  for  a  few  minutes,  even  at  room-temperature,  enzyme 
solutions  are  rapidly  inactivated.  This  action  was  observed  almost  simul- 
taneously by  Signe  and  Sigval  Schmidt-Nielsen  with  rennet, 
Abderhalden  and  Guggenheim  with  tyrosinase,  and  S  h  a  k  1  e  e 
and  M  e  1 1  z  e  r  with  pepsin. 

As  was  formerly  assumed  by  the  author,  inactivation  by  shaking  or 
denaturation  and  skin-formation  are  due  partly  to  a  surface-action  and  partly 
to  the  influence  of  atmospheric  oxygen;  with  many  colloidal  solutions,  shaking 
in  the  air  produces  flocculation  (S.  and  S.  Schmidt-Nielsen,  H., 
1910,  68,  317). 

"According  to  Shaklee  (Zentralbl.  f.  Physiol.,  1909,  23,  4),  at  37° 
pepsin  loses  its  activity  according  to  the  formula  for  bimolecular  reactions 


at  a  —  x 

where  a  is  the  original  amount  of  pepsin  and  x  the  amount  changed  (destroyed) 
in  time  t.     After  12  days,  86%  of  the  enzyme  is  destroyed. 
3  Calculated  by  Arrhenius  (Immunochemistry,  p.  98). 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION      237 

the  previous  treatment  and  especially  on  the  moisture-content 
of  the  enzyme-preparations,  and  they  have  a  real  significance 
only  if  the  compositions  of  the  preparations  are  suitably  denned. 

The  velocity  of  decomposition  of  dissolved  enzymes  and 
its  temperature-coefficient  are  also  largely  dependent  on  the  other 
substances  present  in  the  solution.  Small  proportions  of  acids  or 
bases  often  influence  the  stability  enormously;  thus,  for  example, 
bases  occasion  a  very  considerable  acceleration  of  the  destruction 
of  rennet  and  of  trypsin  (Arrhenius,  Immunochemistry, 
p.  88). 

A  very  complete  study  of  the  influence  of  acids  and  alkalies 
on  the  destruction  of  invertase  has  been  made  by  Hudson 
and  Paine  (Journ.  Amer.  Chem.  Soc.,  1910,  32,  985),  from 
whose  paper  the  following  figure  (p.  238)  is  taken. 

An  extract  from  Table  2  of  Hudson  and  P  a  i  n  e  '  s 
paper  shows  that  at  0-45°,  the  temperature-coefficient  of  the 
destruction  by  acid  or  alkali  does  not  differ  from  that  of  ordi- 
nary reactions.  This  is  very  remarkable,  for,  at  the  optimal  sta- 
bility (H'=  about  10~5),  the  temperature-coefficient  of  enzyme- 
destruction  is,  at  any  rate  between  55  and  65°,  extremely  high. 
It  recalls  the  temperature-coefficients  of  the  denaturation  of  pro- 
teins recently  measured  by  Martin,  and  it  is  to  be  supposed 
that  with  the  enzymes  it  is  a  case  of  similar  denaturation,  i.e., 
of  a  change  of  their  state  of  solution.  In  this  connection,  an 
ultramicroscopical  investigation  would  be  of  interest. 

In  many  cases,  salts  produced  a  marked  increase  in  the  stability 
of  enzymes;  thus,  according  to  V  e  r  n  o  n  (Journ.  of  Physiol.,* 
1901,  27,  174),  the  optimum  of  pancreas-diastase  in  a  starch 
solution  containing  0  •  2%  of  sodium  chloride  is  at  50°,  whilst  in 
pure  aqueous  starch  solution  the  optimum  temperature  is  35°. 

Apart  from  this,  it  is  known  that  many  neutral  substances, 
particularly  proteins  l  and  other  colloids  (Journ.  of  Physiol.,  1904, 
31,  346),  but  more  especially  the  specific  substrates  and  reaction- 
products,  increase  the  stability  of  the  enzymes  to  a  marked  extent. 
This  latter  fact  was  pointed  out  by  O  '  Sullivan  and  T  o  m  p  - 
son  (loc.  cit.).  Biernacki  (Zeitschr.  f.  BioL,  1891, 
28,  49)  and  Vernon  (Journ.  of  Physiol.,  1901,27,  269;  1902, 
28,  375,  448;  1904,  31,  346)  made  the  same  observation  in  the 

1  Non-specifically  hydrolysable  proteins  often  act  as  "buffers." 


238 


GENERAL  CHEMISTRY  OF  THE  -ENZYMES 


case  of  trypsin,1  and  W  o  h  1  and  G  1  i  m  m  (Biochem.  Z.,  1910, 
27,  365)  in  that  of  amylase.  In  other  instances,  for  example, 
according  to  the  author's  measurements,  with  invertase,  the 
protective  action  of  the  substrate  is  slight.  The  stability  of 
this  enzyme  is,  however,  considerably  increased  by  the  presence 


.10 


.08  .06  .04  .02 

Concentration  of  Acid-< — 


.02  .04  .06  .08 

->  Concentration  of  Alkali 


.10 


FIG.  7. 


of   fructose    (Hudson 
Soc.,  1911,  32,  988). 


and   Paine,    Journ.    Amer.    Chem. 


1  In  this  connection  mention  must  be  made  of  the  observations  of  W  . 
Cramer  and  Beam  (Proceedings  of  the  Physiol.  Soc.,  June  2,  1906; 
see  Journ.  of  Physiol.,  1906,  34,  XXXVI),  according  to  whom  active  pepsin 
is  retarded  by  the  addition  of  pepsin  solutions  inactivated  at  60°,  whereas 
preparations  inactivated  at  100°  produce  little  or  no  retardation. 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION      239 

In  dilute  solution  diastase  keeps  better  than  in  more  concen- 
trated ones  (E  f  f  r  o  n  t ,  Enzymes  and  their  Applications, 
1902,  p.  56). 

The  t  emper  atur  e- coef  f  i  c  i  ent  s  of  the  enzy- 
mic  reactions  themselves,  that  is,  the  alterations 
of  the  velocity-constants  k  with  the  temperature  are  of  the  same 
order  of  magnitude  as  those  of  other  chemical  processes.  An 
attempt  should  always  be  made  to  measure  the  temperature- 
coefficient  in  a  region  of  temperature  where  the  destruction  of 
the  enzyme  comes  into  consideration  as  little  as  possible.  In 
most  of  the  previous  measurements,  the  distance  from  the  op- 
timum is  so  slight  that  the  constants  [i  are  influenced  by  the 
destruction  of  the  enzyme  and  are  consequently  too  low.  Fur- 
ther, as  the  following  examples  show,  the  errors  of  observation 
are  generally  very  large,  even  in  the  work  of  reliable  experimenters. 

A  u  1  d  (Journ.  Chem.  Soc.,  1908,  93,  1275) :  measurements 
on  amygdalin-emulsin. 


kv,  :/Ci5  =  2-37 
/c3o  :fc20  =  l-81 
£35  :  &25  =2-14 
fc40  :/c30=l-68 


£45 

&50 


K  a  s  1 1  e  and  Loevenhart  (Amer.  Chem.  Journ.,  ]  900> 
24,  501)  left  tubes  containing  4  c.c.  of  water,  0-1  c.c.  of  toluene 
and  1  c.c.  of  a  10%  liver-  or  pancreas-extract  for  5  minutes  in 
baths  at  40°,  30°,  20°,  10°,  0°  and  - 10°,  so  that  they  assumed 
these  temperatures.  Ethyl  butyrate  (0-26  c.c.)  was  then  added 
and  the  solutions  titrated  after  30  minutes. 


Percentage  hydrolysed. 

By  liver-extract. 

By  pancreas-extract. 

40° 

ll-29(?) 

2-82 

30 

5-96 

3-16 

20 

5-27 

2-51 

10 

3-89 

1-88 

0 

2-26 

1-25 

-10 

0-70 

240  GENERAL  CHEMISTRY   OF  THE  ENZYMES 

The  value  obtained  with  liver-extract  at  40°  must  be  due  to 
an  error  of  experiment.  H  a  n  r  i  o  t  (C.  R.,  1897,  124,  778) 
obtained  similar  results  with  his  esterases  from  serum  and  pancreas. 

Reference  must  finally  be  made  to  a  series  of  experiments 
made  by  C  h  o  d  a  t  (Arch.  Sci.  phys.  nat.,  1907,  23,  13)  on  the 
action  of  tyrosinase  on  tyrosine. 

The  second  row  below  gives  the  times  in  which  the  solution 
had  attained  a  certain  intensity  of  colour. 

Temperature 0°       10°     20°     30°     40°     45°     50° 

Time  (minutes) 180       100      60      40      30      20       10 

Very  unreliable  quantitatively  are  the  temperatures  given  by 
Tammann  (loc.  cit.)  for  invertase  and  cane-sugar, 
Lindner  and  Krober  (Chem.  Ber.,  1895,  28,  1053)  for 
maltase,  H  a  n  r  i  o  t  and  Camus  (C.  R.,  1897,  124,  235) 
for  serum-esterase  and  monobutyrin,  M  i  q  u  e  1  (see  H  e  r  - 
z  o  g  ,  1  o  c  .  c  i  t .)  for  urease,  and  by  experiments  made  accord- 
ing to  M  e  1 1 '  s  method. 

Consequently  reference  will  only  be  made  to  the  calculation 
of  these  results  by  H  e  r  z  o  g  (Zeitschr.  f.  allg.  Physiol.,  1904, 
4,  189). 

In  the  following  table  (p.  241)  are  collected  the  data  as  yet 
obtained  concerning  the  temperature-coefficients  of  enzyme- 
reactions.1 

In  other  cases  the  value  of  [JL  changes  considerably  with 
temperature;  this  is  shown,  for  instance,  byMuller-Thur- 
gau's  results  with  amylase  (Landw.  Jahrb.,  18.85,  795). 

An  extended  investigation  of  the  temperature  coefficients 
of  alcoholic  fermentation  by  living  yeast  has  been  made  by 
S  1  a  t  o  r  ,  with  the  following  results : 

t  vt+io  :  vt 

5°  5-6 

10  3-8 

15  2-8 

20  2-25 

25  1-95 

30  1-60 

1  Complicated  biological  processes,  such  as  the  assimilation  of  carbon  by 
green  leaves,  are  not  considered  here. 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION       241 


Author. 

Substrate  and  enzyme. 

Temp.- 
interval. 

kt+io 
kt 

(A 

K  a  s  1  1  e     and     Loevenhart 
(1  o  c  .  c  i  t  .)        

Ethyl  butyrate,  esterase 

20-30° 

1-3 

4,650 

Tammann   (loc.  cit.)  
K  j  e  1  d  a  h  1  (Medd.  fra  Carlsberg 
Lab     1881    335)                

Cane-sugar,  invertase 
Cane-sugar,  invertase 

20-30 
30-40 

1-4 
1-5 

6,000 
7;800 

O  '  S  u  1  1  i  v  a  n  and  T  o  m  p  s  o  n 
(loc      cit)                  

Cane-sugar,  invertase 

40-50 

1-4 

6,800 

S  e  n  t  e  r     (Zeitschr.    f.    physikal. 
Chem.,  1903,  44,  257)  
Chodat   (loc     cit  ) 

HzOa,  catalase 

0-10 
20-30 

1-5 
1-5 

6,200 

7  200 

E  u  1  e  r  and  af   Ugglas   (loc. 
cit)                                     

0-20 

2-0 

11,000 

Vernon     (Journ.     of     Physiol., 
1901,  27,  190)  

Starch,  amylase 

20-30 

2-0 

12,300 

Vernon    (                ibid.                  ) 
Vernon   (ibid.  1903,  30,  364).  .  . 
Tammann   (loc.   cit.)  
Vernon     (Journ.    of   Physiol., 
1903,  30,  364)  
Taylor    (Journ.  of  Biol.    Chem., 
1906,  2   87) 

Milk,  rennet 
Witte's  peptone,  trypsin 
Salicin,  emulsin 

Witte's  peptone,  erepsin 
Triacetin,  lipase 

20-30 
15-25 
15-25 

15-25 
18-28 

2-1 
2-3 
2-4 

2-6 
2-6 

13,400 
14,300 
15,000 

16,400 
16  700 

F  u  1  d   (Hofm.  Beitr.,  1908,  2,  169) 
B  a  y  1  i  s  s     (Arch.    Sci.    Biol.,    St. 
Petersburg,    1904,   11,  261,  Sup- 
plement)  

Milk,  rennet 
Casein,  trypsin 

30-40 
20-7-30-7 

3-2 
5-3 

22,000 
37  500 

Heterogeneous     systems 


A  b  e  r  s  o  n       (Rec.     Trav.     Chim. 
Pays-Bas,  1903,  22,  100)  

Sugar,  living  yeast 
Sugar,  permanent  yeast 

18-28° 
15-25 

2-7 

2-8 

15,600 
18,000 

Herzog   (H.,  1902,37,  160).. 

... 

In  consequence  of  the  foregoing  data  it  must  again  be 
emphasised  that  the  influence  of  temperature  on  enzymic  reac- 
tions is  exactly  denned  only  by  determination  of  the  inactivation 
constant  kE  at  a  given  temperature  and  measurement  of  the 
initial  velocities  of  the  reaction  itself  at  temperatures  at  least 
20°  lower  than  the  above. 

The  following  very  instructive  numbers,  obtained  by  G  e  r  - 
ber  (Soc.  Biol.,  1903,  63,  375),  show  how  the  temperature- 
coefficient  of  an  enzymic  reaction  may  depend  on  the  quantity 
of  enzyme  present: 


242 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Concen- 

Time  of  coagulation  of  milk  at  different  temperatures. 

rennet. 

25° 

30° 

33° 

36° 

39° 

42° 

45° 

0-005 
0-010 

30'  20" 
14  45 

29'  00" 
11   30 

(  no  coag. 
\  in  360' 

r  oo" 

1  no  coag. 
in  360' 

(no  coag. 
in  360' 

No 

No 

0-015 

9  40 

7  25 

4     40 

5'    35" 

( 

coag. 

coag. 

0-020 

7  30 

5  00 

2     30 

3     15 

5'   30" 

0-025 

6  15 

3  30 

2    05 

2     20 

2     40 

in 

0-030 

4  40 

2  50 

1     40 

1     30 

1     40 

360' 

in 

0-040 

3  40 

2  20 

1     30 

1     00 

0     50 

0-050 

3  00 

1   50 

1     10 

0     55 

0     40 

2'   05" 

360' 

0-075 

2  20 

1   20 

0     50 

0    40 

0     30 

1     00 

0-100 

1  40 

1   00 

0    40 

0     30 

0     25 

0    35 

0'   45" 

Very  striking  is  the  result  of  a  comparison  between  the  values 
of  [L  for  the  acceleration  of  the  inversion  of  cane-sugar  by  an  acid 
and  by  invertase  respectively;  the  data  on  pp.  231  and  241 
give: 

Cane-sugar-hydrochloric  acid.  Cane-sugar-invertase. 

H  =  25,600  [1  =  11,000 

Although  enzymic  temperature-coefficients  had  been  deter- 
mined by  very  reliable  experimenters  (Kjeldahl,  T  a  m  - 
m  a  n  n  ,  O  '  Sullivan  and  Tompson),  new  investiga- 
tions on  this  point  were  to  be  desired.  On  this  account,  the 
author  and  Miss  B.  af  Ugglas  have  made  fresh  determina- 
tions of  these  coefficients  under  various  experimental  conditions 
(H.,  1910,  65,  124);  at  the  optimum  concentration  of  hydrogen- 
ions,  it  was  found  that  /CSQ  :  £20  =  1-9 -2-1.  This  is  in  agree- 
ment with  the  result  obtained  by  V  i  s  s  e  r  (Zeitschr.  f.  physikal. 
Chem.,  1905,  52,  257),  namely  2,  for  the  ratio  k2o  :  kio. 

The  first  conclusion  that  could  be  drawn  is  that  the  con- 
stant [L  includes  the  heat-change  occurring  during  the  forma- 
tion of  the  compound  between  the  cane-sugar  and  the  acid  or 
invertase.  It  is  also  possible  that  the  small  rise  of  temperature 
taking  place  during  enzymic  inversion  may  be  due  to  rise  of  tem- 
perature not  only  irreversibly  destroying  the  invertase  but  also 
reversibly  inactivating  it;  apart  from  the  destroyed  portion, 
the  invertase  resumes  its  original  activity  at  the  lower  temperature. 
Hence,  rise  of  temperature  renders  the  substrates,  cane-sugar 


INFLUENCE  OF  TEMPERATURE  AND   RADIATION       243 

and  water,  more  active  and  the  catalysing  enzyme  less  active. 
Other  reactions,  for  instance,  the  hydrolysis  of  esters,  also  exhibit 
smaller  temperature-coefficients  for  enzymic  than  for  acid  catal- 
ysis. For  non-enzymic  reactions,  S  1  a  t  o  r  (Zeitschr.  f . 
physikal.  Chem.,  1903,  45,  547)  found  temperature-coefficients 
varying  with  the  catalyst. 

Very  ill-defined  is  the  so-called  "  optimum  temperature," 
the  position  of  which  depends  entirely  on  the  period  or  phase 
of  the  reaction  considered.  Indeed,  even  at  the  optimal  tem- 
perature the  enzyme  undergoes  partial  destruction  during  the 
reaction,  so  that  if  comparison  is  made  of  the  times  taken  for  the 
reaction  to  proceed  to  the  extent  of  one-half,  the  optimum  is 
apparently  lower  than  if  only  the  first  one-fifth  of  the  reaction 
is  considered.  The  real  initial  velocity  will,  in  general,  show  no 
optimum  if  the  time  of  observation  is  made  short  enough.  For 
practical  purposes  it  may  be  of  interest  to  know  the  temperature 
at  which  the  reaction  proceeds  most  rapidly,  and  it  would  then 
be  best  to  consider  the  times  in  which,  say,  90-95%  of  the  sub- 
strate is  decomposed.  In  any  case,  in  giving  the  optimum  tem- 
perature, it  must  be  stated  for  which  stage  of  the  reaction  it 
holds. 

Still  more  indefinite  are  most  of  the  data  on  "maximum 
temperatures"  (temperatures  of  destruction).  Measure- 
ments of  these  temperatures  are  of  value  only  when  the  duration 
of  the  experiment  and  the  magnitude  of  the  weakening  are  deter- 
mined. It  is  therefore  advisable  to  give  that  temperature  at 
which  the  enzyme  is  weakened,  for  example,  to  the  extent  of  one- 
half  in  30  minutes;  still  better  is  it  to  measure  the  inactivation 
constant  ks  of  the  dissolved  enzyme  at  a  given  temperature. 

The  presence  and  concentration  of  other  substances  in  the 
solution  may  influence  both  the  optimal  and  the  maximal  tem- 
peratures very  considerably;  marked  alterations  of  these  tem- 
peratures are  produced  especially  by  acids  and  bases,  so  that  in 
measurements  of  the  stability  it  is  necessary  to  define  the  con- 
centration of  the  H'-  or  OH'-ions.  Non-electrolytes — so  long 
as  they  do  not  constitute  the  substrate  or  a  product  of  the  reaction 
— appear  to  have  little  influence,  the  stability-constant  being, 
therefore,  readily  reproducible  (cf.  the  measurements  of  B.  a  f 
U  g  g  1  a  s  and  Kullberg,  loc.  cit.). 

The  very  great  differences  existing,  according  toR.Huerre 


244  GENERAL  CHEMISTRY   OF  THE  ENZYMES 

(C.  R.,  1909,  148,  300),  between  the  maltase  of  white  maize 
and  that  of  yellow  maize,  may  be  due  to  the  action  of  a  foreign 
substance  of  some  kind;  but  it  can,  by  no  means,  be  denied  that 
the  two  sorts  of  maize  may  contain  different  maltases. 

For  most  enzymes  the  optimal  temperature  is  stated  to  be 
between  37  and  53°,  and  the  maximal  temperature  between  60° 
and  75°. 

Many  oxydases  resist  surprisingly  high  degrees  of  tem- 
perature. That  known  as  Medicago  -laccase  is  especially 
stable  to  heat,  as  is  to  be  expected  from  its  composition  (see  p.  62). 
Other  oxydases  are  destroyed  only  at  80-90°  (K  a  s  1 1  e  ,  Chem. 
ZentralbL,  1906,  77,  i,  1554). 

Also  peroxydases,  at  any  rate  in  the  natural  juices,  are  only 
slightly  injured  by  heating.  Thus,  a  preliminary  experiment 
with  the  juice  from  pressed  horseradish  showed  that  heating 
for  two  hours  at  60°  diminished  the  activity  of  the  peroxydase 
on  guaiaconic  acid  only  in  the  proportion  of  7  :  5.  For  ^  the 
very  low  value,  4000,  was  obtained. 

Apparently  still  more  resistant  is  myrosin,  which,  according 
to  Guignard's  experiments  (C.  R.,  1890,  111,  249,  920; 
Bull.  Soc.  Bot.  de  France,  1894,  [3],  418),  is  not  destroyed  even 
at  81°,  although  a  knowledge  of  the  duration  of  the  heating  is  to 
be  desired. 

Peculiar  behaviour  towards  high  temperatures  has  been 
observed  byDelezenne,  Mouton  and  P  o  z  e  r  s  k  i 
(Soc.  Biol.,  1906,  60,  68,  390)  in  the  case  of  papain.  At  tem- 
peratures up  to  40°,  papain  exerts  no  digestive  action  on  egg- 
albumin  or  blood-serum.  Digestion  only  occurs,  and  then  very 
rapidly,  on  further  heating  of  the  solution.  These  results  were 
completely  confirmed  byJonescu  (Biochem.  Z.,  1906,  2,  177), 
who  studied  the  differences  between  ordinary  and  "  heat- 
digestion,"  while  Gerber  (Soc.  Biol.,  1909,  66,  227)  has 
also  remarked  the  notable  resistance  of  papain  to  high 
temperatures. 

Towards  low  temperatures,  enzymes  appear  to  be  highly 
resistant;  thus,  Miss  White  showed  that  the  enzymes  of 
cereals  are  not  destroyed  by  exposure  to  the  temperature  of 
liquid  air  for  two  days  (Proc.  Roy.  Soc.,  £.,  1909,  81,  440). 

To  sum  up,  the  sensitiveness  of  enzymes  to  heat  is,  indeed, 
very  great,  but  is  not  so  marked  as  with  the  toxines,  for  which 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION      245 

M  a  d  s  e  n     found    the    temperature-constants    of    spontaneous 
decomposition  in  solution  to  be  as  high  as  [L  =  198,500. 

Enzymic  reactions  appear  to  have  rather  lower  temperature- 
coefficients  than  the  corresponding  non-enzymic  catalyses. 

INFLUENCE   OF  RADIATION 

The  manifold  action  exerted  by  light  on  the  processes  occurring 
in  living  cells  and  tissues  has  naturally  given  rise  to  the  impres- 
sion that  enzymes  are  sensitive  to  rays  of  various  wave-lengths. 
The  success  of  the  modern  photo-therapeutic  methods  of 
F  i  n  s  e  n  and  others,  on  the  one  hand,  and  the  knowledge  that 
toxines  undergo  very  rapid  destruction  in  the  light,  on  the  other, 
endow  this  subject  with  considerable  practical  and  scientific 
interest. 

1.  Light-rays 

It  may  be  mentioned  firstly  that,  in  general,  enzymes  do  not 
appear  to  exhibit  so  high  a  degree  of  sensitiveness  to  light  as 
do  the  toxines.  This  observation  was,  indeed,  made  some  years 
ago  by  O  .  E  m  m  e  r  1  i  n  g  (Chem.  Ber.,  1901,  34,  3811). 

Hertel  subjected  a  number  of  enzymes  and  toxines  to 
the  influence  of  light-rays  and  observed,  among  other  results, 
that  trypsin  and  also  diastase  and  rennet  are  weakened  by  rays 
having  the  wave-length  280  ^.  His  investigations  also  showed 
that  the  destruction  of  enzymes  requires  a  longer  exposure  than 
does  that  of  the  toxines,  the  enzymes  being  therefore  decidedly 
more  photo-stable  substances  than  the  toxines  (Biol.  Zentralbl., 
1907,  27,  510). 

Just  as  in  the  study  of  the  thermo-lability  of  the  enzymes, 
so  also  in  investigating  the  action  of  light,  a  distinction  must 
be  drawn  between  the  destruction  of  the  enzyme  by  light  and 
the  alteration  of  the  enzymic  action  under  the  influence  of  the 
radiation.  In  so  far  as  the  results  already  obtained  indicate, 
the  former  influence  predominates,  the  observed  retardations 
of  enzyme-action  being  therefore  due  principally  to  a  partial 
annihilation  of  the  enzyme  molecule. 

In  his  paper  cited  above,  E  m  m  e  r  1  i  n  g  states  that,  in 
absence  of  air,  invertase,  lactase,  emulsin,  amylase,  trypsin  1 

1  According  to  Fermi  and  P  e  r  n  o  s  s  i  (Zeitschr.  f .  Hygiene,  1894, 
18,  83),  pepsin  and  trypsin  are  weakened  in  sunlight. 


246  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

and  pepsin  are  injured  but  slightly  by  daylight;  yeast  maltase 
and  rennet  are  rather  more  sensitive  to  these  conditions.  The 
activity  of  the  last-named  enzyme,  in  1%  aqueous  solution, 
was  reduced  to  one-half  by  diffused  sunlight,  and  to  one-third 
by  direct  sunlight,  in  the  course  of  five  days. 

D  o  w  n  e  s  and  Blunt  found  this  action  of  light  to  be 
of  slight  extent  and  the  same  result  was  obtained  by  F.  W  e  i  s 
(Medd.  fra  Carlsberg  Lab.,  1903,  5,  135)  with  the  proteolytic 
enzyme  of  malt  and  by  Schmidt-Nielsen  (Medd.  fra 
Finsens  med.  Lysinstitut,  1903)  with  chymosin.  On  the  other 
hand,  F.  G  .  Kohl  (Beitr.  z.  Bot.  Zentralbl.,  1908,  23,  64) 
states  that  invertase  is  considerably  affected  even  by  diffused 
daylight.  The  disagreement  between  this  statement  and  the 
earlier  observations  is  explained  by  the  results  of  J  a  m  a  d  a  and 
Jodlbauer  (Biochem.  Z.,  1908,  8,  61),  who  showed  that  the 
rays  of  sunlight  which  pass  through  glass  are  alone  capable  of 
injuring  invertase,  but  to  a  marked  extent  only 
in  presence  of  oxygen. 

With  reference  to  the  stability  of  catalase  under  the  action 
of  light,  a  comprehensive  investigation  was  carried  out  by 
Lockemann,  Thies  and  W  i  c  h  e  r  n  (H.,  1909,  58, 
390).  The  inhibiting  action  of  light  on  blood-catalase,  both 
when  the  blood-solution  is  kept  and  during  the  reaction  with 
hydrogen  peroxide,  is  greatest  with  white  and  least  with  red  light, 
blue  occupying  an  intermediate  position.1  Also  in  this  case, 
according  to  Z  e  1 1  e  r  and  Jodlbauer  (Biochem.  Z.,  1908, 
8,  84),  appreciable  injury  is  produced  by  visible  rays  only  when 
oxygen  is  present;  a  similar  result  was  arrived  at  by  Z  e  1 1  e  r 
and  Jodlbauer,  and  almost  simultaneously  by  Bach 
(Chem.  Ber.,  1908,  41,  225),  with  peroxydase. 

The  results  of  a  large  number  of  investigations  are  in  agree- 
ment in  indicating  that  the  inhibiting  influence  of  ultra- 
violet rays  is  much  greater  than  that  of  the  visible  rays. 
The  action  of  these  rays  was  investigated  firstly  and  very  com- 

1  In  absence  of  catalase,  additions  of  sodium  chloride  retard  the  decom- 
position of  hydrogen  peroxide  by  light.  For  the  sensitiveness  to  light  of 
solutions  of  the  peroxide  either  containing  or  free  from  catalase,  W  o  . 
O  s  t  w  a  1  d  (Biochem.  Z.,  1908,  10,  1)  found  the  influences  of  different 
kinds  of  light  to  be  in  the  following  order  of  diminishing  magnitude:  white, 
violet,  yellow,  dark. 


INFLUENCE  OF  TEMPERATURE  AND  RADIATION      247 


pletely  by  Reynolds  Green  (Trans.  Roy.  Soc.,  1897, 
188,  167),  who  showed  that  violet  and  ultra-violet  rays  destroy 
diastase,  but  that  the  action  of  this  enzyme_is  enhanced  by  visible 
rays  owing  to  activation  of  the  zymogen.  As  was  mentioned 
above,  visible  rays  have  only  a  slight  retarding  action  on  chymosin, 
catalase  and  peroxydase,  but  these  enzymes  are  rapidly  and  per- 
manently inactivated  by  ultra-violet  radiation  (Schmidt- 
Nielsen,  Zeller  and  Jodlbauer).  Signe  and  Sigval 
Schmidt-Nielsen  made  a  detailed,  kinetic  study  of  the 
destruction  of  rennet  by  ultra-violet  light  (H.,  1908,  58,  235), 
whilst  shortly  beforehand  Georges  Dreyer  and  O  1  a v 
H  a  n  s  s  e  n  (C.  R.,  1907,  145,  564)  showed  that  the  destruc- 
tion of  enzymes  by  radiation  follows  the  law  for  unimolecular 
reactions. 

Of  Schmidt-Nielsen's  experiments,  which  were 
made  in  the  Finsen  Institute  with  a  mercury-vapour  lamp,  the 
following  may  be  described : 

A  1%  solution  of  dry,  commercial  rennet  powder  was  exposed 
to  the  radiation  for  a  definite  period  and  the  time  required  for 
the  coagulation  of  cow's  milk  subsequently  measured;  this 
time  was  assumed  to  be  a  direct  measure  of  the  amount  of  unaltered 
enzyme  in  the  solution. 


Temperature, 
°C. 

Exposure  to  the  light, 
minutes. 

Time  of  clotting, 
minutes. 

1000/t. 

24-2 

0 

8-5 



24-2 

1-0 

23-5 

442 

24-2 

1-5 

39-25 

443 

24-2 

2-0 

71-0 

461 

0 

0 

7-7 



12-75 

1-0 

19-5 

405 

12-80 

1-5 

34-5 

434 

12-85 

2-0 

59-0 

442 

13-90 

2-0 

56-0 

431 

12-95 

2-0 

54-5 

425 

As  was,  indeed,  to  be  expected,  these  numbers  show  that 
the  destruction  of  rennet  by  light  is  undoubtedly  a  unimolecular 
reaction.  But  what  deserves  special  attention  is  the  extraor- 
dinarily small  temperature-coefficient  of  this  reaction.  That  such 


248  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

processes  possess  low  temperature-coefficients  has  been  repeatedly 
observed  and  seems  to  be  a  general  rule.  It  may  be  assumed 
in  the  above  case  that  the  temperature  of  the  chymosin-mole- 
cules,  after  exposure  to  the  light,  is  considerably  higher  than  that 
of  the  surrounding  solution  and  is  not  very  different  in  the  two 
series  of  experiments;  the  temperature  of  the  solution  had  therefore 
little  influence  on  the  thermal  condition  of  the  chymosin. 

In  Schmidt-Nielsen's  experiments,  the  reaction 
constant  diminished  with  increase  of  the  concentration.  This 
is  not  surprising,  since  the  destruction  by  heat  of  enzymes  proceeds 
more  slowly  in  their  concentrated  than  in  their  dilute  solutions. 
Hence,  as  their  concentration  increases,  enzymes  become  more 
stable  to  both  heat  and  light.  Of  the  total  effect  of  the  radia- 
tion of  the  mercury  lamp,  96%  is  due  to  rays  with  the  wave- 
lengths 220-250  \L[L  and  only  about  0-3%  to  the  visible  rays. 

Very  interesting  experiments  have  been  made  byvonTap- 
p  e  i  n  e  r  and  his  co-workers  on  the  action  of  sunlight  on  dias- 
tases and  invertase  in  presence  of  fluorescent  substances  (sensi- 
tisers).  Very  small  quantities  of  eosin,  Magdala  red  or  quinoline 
red  are  sufficient  to  cause  sunlight — which  of  itself  is  with  action — 
to  exert  a  marked  inhibiting  effect. 

In  diffuse  daylight  the  fermentative  power  of  yeast  is  destroyed 
by  fluorescent  bodies.  With  living  yeast,  only  certain  fluorescent 
substances  are  active;  but  with  permanent  acetone-yeast  and, 
still  more,  with  yeast-juice,  all  the  fluorescent  bodies  examined, 
such  as  eosin,  methylene  blue,  fluorescein,  dichloranthracene- 
disulphonic  acid,  etc.,  induce  considerable  diminution  of  the 
fermentative  power  (von  Tappeiner,  Biochem.  Z.,  1908, 
8,  47). 

Also  on  catalase  all  the  fluorescent  substances  investigated 
have  a  sensitising  action,  whilst  with  peroxydase  this  is  the  case 
only  with  eosin  and  Bengal  red;  in  both  these  instances,  the 
action  only  occurs  when  the  ultra-violet  rays  are,  as  far  as  possible, 
lacking  ( J  a  m  a  d  a  and  Jodlbauer,  Biochem.  Z.,  1908, 
8,  61 ;  Z  e  1 1  e  r  and  Jodlbauer,  ibid.,  84;  Karamit- 
s  a  s  ,  Dissertation,  Munich,  1907). 

Thus  the  biological  action  of  light  is  of  two  kinds  (Jodl- 
bauer and  von  Tappeiner,  Deut.  Arch.  f.  klin.  Med., 
1906,  85,  386):  One  requiring  the  presence  of  oxygen  and 
accelerated  by  fluorescent  substances,  and  the  other  produced 


INFLUENCE   OF  TEMPERATURE  AND  RADIATION      249 

only  by  ultra-violet  rays  without  any  part  being  played  by 
oxygen  or  fluorescent  substances. 

The  results  mentioned  above  show  that  light  exerts  actions 
of  two  kinds  on  enzymes : 

(1)  A    destroying    action,    corresponding   with    denaturation 
by  heat. 

(2)  An  activating  effect,  due  to  conversion  of  the  "  zymogen  " 
into  the  active  enzyme. 

2.  Other    Forms   of   Radiation 

By  Rontgen  rays,  enzymes  are  not  weakened.  This 
was  shown  by  P.  F.  R  i  c  h  t  e  r  and  Gerhartz  (Berl.  klin. 
Wochens.,  1908,  45,  646)  to  be  the  case  with  chymosin,  yeast, 
pepsin,  pancreatin  and  papain,  while  Lockemann,  Thies 
and  W  i  c  h  e  r  n  obtained  the  same  result  with  blood-catalase. 

Radium  rays  and  radium  emanation,  how- 
ever, do  appear  to  exert  an  action  on  enzymes,  although,  accord- 
ing to  W  i  1  c  o  c  k  (Journ.  of  Physiol.,  1907,  34,  207),  tyrosinase 
is  not  affected  by  radium  rays,  while  Schmidt-Nielsen 
found  that  even  a  very  active  preparation  of  radium  has  a  very 
slight  effect  on  chymosin;  Henri  and  Mayer  (C.  R.,  1904, 
138,  521)  state  that  invertase,  emulsin  and  trypsin  are  injured. 
Against  these  negative  assertions  are  arrayed  a  number  of  other 
positive  results. 

B  e  r  g  e  1 1  and  B  i  c  k  e  1  (Verhandl.  d.  Kongr.  f.  inn.  Med., 
Wiesbaden,  1906)  first  showed  that  peptic  digestion  is  favoured 
by  the  emanation.  N  e  u  b  e  r  g  (Verhandl.  d.  deutsch.  path. 
Ges.,  1904)  and  Wohlgemuth  (ibid)  observed  accelera- 
tion of  the  autolytic  processes  by  radium  radiation,  while 
Loewenthal  and  E  d  e  1  s  t  e  i  n  (Biochem.  Z.,  1908,  14, 
484)  found  these  processes  to  be  facilitated  by  the  emanation. 
Also  Loewenthal  and  Wohlgemuth  (Biochem.  Z., 
1909,  21,  476)  have  recently  proved  that  radium  emanation  is 
capable  of  accelerating  the  action  of  the  diastatic  enzyme  of  the 
blood,  liver,  saliva  or  pancreas.  "  This  favourable  action  is 
not  always  observable  immediately;  very  often  retardation 
occurs  during  the  first  24  hours,  this  being  gradually  neutralised 
and  then  replaced,  if  the  experiment  is  sufficiently  prolonged, 
by  an  acceleration.  In  other  cases,  the  emanation  produced  only 


250  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

inhibition,  which  was  not  compensated  when  the  duration  of  the 
experiment  was  extended." 

Acceleration  of  enzyme-action  by  the  emanation  has  been 
established  with  pepsin  and  trypsin. 

Of  importance  from  a  therapeutic  standpoint  isGudzent's 
discovery  that  the  enzyme  of  purine-metabolism  is  activated 
by  radium  emanation. 

Action  ofmesothorium. 

The  mesothorium  bromide  discovered  by  0.  H  a  h  n  emits 
rays  of  three  kinds:  a-,  @-  and  y-rays.  Of  these,  the  @-  and  y- 
rays  pass  without  alteration  through  a  mica  plate,  if  this  is  not 
too  thick;  they  are  also  able  to  traverse  a  thin  sheet  of  glass,  but 
are  then  weakened  to  some  extent.  B  i  c  k  e  1  and  M  i  n  a  m  i 
(Berl.  klin.  Wochens.,  1911,  48,  1413)  found  that  exposure  of 
carcinoma,  sarcoma  and  liver  to  the  radiation  of  mesothorium 
bromide — the  action  of  emanation  or  a-rays  being  excluded — 
has  no  influence  on  the  autolytic  enzymes.  These  authors  regard 
their  results  as  of  fundamental  importance.  If  it  is  a  fact  that  the 
P-  and  y-rays  of  mesothorium  are  identical  in  every  respect  with 
the  @-  and  y-rays  of  radium,  it  must  be  concluded  that  the  activa- 
tion of  the  autolytic  enzymes  observed  as  a  result  of  the  action 
of  radium  is  s  o  1  e  1  y  an  effect  of  the  a-rays  or  emanation.  The 
same  is  probably  the  case  with  other  enzymes.  According  to 
Minami  (Berl.  klin.  Wochens.,  1911,  48,  1798)  the  £-  and 
y-rays  of  mesothorium  exert  a  very  slight  influence  on  the 
digestive  enzymes,  amylase,  pepsin  and  trypsin.  The  biological 
action  of  thorium  emanation  has  been  studied  by  B  i  c  k  e  1 
(Berl.  klin.  Wochens.,  1911,  48,  2107) ;  like  that  of  radium  emana- 
tion, it  consists  sometimes  of  a  retardation  and  sometimes  of  an 
activation  of  enzymic  action,  and  is  more  intense  than  that  of 
g-  and  y-rays. 

In  general,  it  may  be  stated  that  the  healing  action  found 
by  experience  to  be  exerted  by  radium  emanation  depends  on 
the  activation  of  enzymes.  The  promotion  of  plant-growth  by 
the  emanation  (F  a  1 1  a  and  S  c  h  w  a  r  z  ,  Berl.  klin.  Wochens., 
1911,  48)  is  also  to  be  attributed  to  enzyme-activation. 


CHAPTER  VI 
CHEMICAL    STATICS    IN    ENZYME    REACTIONS 

THE  position  of  equilibrium  of  a  chemical  system  is  deter- 
mined, as  is  well  known,  by  the  law  of  mass  action. 

If  1  mol.  of  acetic  acid  reacts  with  1  mol.  of  alcohol,  so  that 
1  mol.  of  ester  and  1  mol.  of  water  are  formed,  then,  according 
to  the  law  of  mass  action- 

[ester] 


[acid]  [alcohol] 

if  the  concentrations  of  the  substances  in  dilute  aqueous  solution 
are  indicated  by  [  ]  and  K  denotes  the  equilibrium  constant. 

According  tovan't  Hoff,  chemical  equilibrium  of  the 
above  reaction  is  due  to  the  equality  of  the  velocity  v\  of  ester- 
formation  and  of  the  velocity  V2  of  ester-decomposition,  so  that, 

v\  =  &i[acid]  [alcohol]  =  V2  =  fetester], 
and  therefore 

[ester]         _T^_&I 
[acid]  [alcohol]  ~     ~  k2' 

The  position  of  equilibrium  is  independent  of  the  rapidity 
with  which  it  is  reached  and  also — as  exact  experiments  show — 
independent  of  the  presence  and  concentration  of  a  catalyst, 
in  so  far  as  this  does  not  combine  to  an  appreciable  extent  with 
the  components  of  the  system. 

As  was  mentioned  in  Chapter  IV  (p.  128),  a  number  of 
investigators  have  arrived  at  the  conclusion  that  enzymic  reactions 
are  effected  by  means  of  a  compound  of  the  enzyme  and  the 
substrate. 

With  the  non-enzymic  hydrolyses  which  have  been  as  yet 
investigated  and  in  which,  according  to  the  author's  theory,  the 

251 


252  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

increase  of  active  molecules  is  due  to  the  formation  of  a  salt  of 
the  substrate  with  the  catalysing  acid,  the  concentration  of  the 
compound  substrate-catalyst  is  so  small  that  no  great  alteration 
occurs  in  the  concentration  of  the  substrate  or  catalyst,  either 
during  the  course  of  the  reaction  or  when  equilibrium  has  been 
attained. 

Enzymic  decompositions  usually  differ  from  those  effected 
by  inorganic  catalysts  in  that  their  velocity  is  determined  not 
only  by  the  absolute  concentration  of  the  catalyst  (enzyme) 
but  also,  and  to  a  far  greater  extent,  by  the  concentration-r  a  t  i  o 
between  enzyme  and  substrate.  If  the  substrate  is  in  excess, 
the  velocity  of  reaction  is  approximately  proportional  to  the 
concentration  of  the  enzyme,  whilst  if  excess  of  the  latter  is  present, 
the  velocity  will  be  very  nearly  proportional  to  the  concentration 
of  the  substance;  in  every  case,  the  velocity  of  reaction  appears 
to  be  proportional  to  the  concentration  of  the  "  intermediate 
product." 

On  the  other  hand,  quantitative  study  of  enzyme-reactions 
has  shown  that  the  products  of  reaction  are  also  fixed  by  the 
enzyme  (Henri,  Bodenstein,  and  others). 

The  question  now  to  be  considered  is  the  relations  in  the 
case  of  enzymic  reactions. 

A  distinction  must  here  be  made  between  true  equilibria 
and  end-states. 


A.    Equilibria 

The  assumption  of  the  existence  of  the  molecules  enzyme- 
substrate  and  enzyme-reaction  product  which  bring  about  the 
reaction  presumes  that  the  mutual  action  between  these  mole- 
cules proceeds  far  more  rapidly  than  those  between  the  free 
substrate  molecules  and  their  decomposition  products.  It  is, 
hence,  principally  the  concentrations  of  the  molecules  of  the 
enzyme-substrate  and  enzyme-reaction  product  which  condi- 
tion the  end-state. 

The  simple  assumption  may  first  be  made  that,  in  miitMime, 
equal  numbers  of  enzyme-substrate  and  enzyme-reaction  pro- 
duct molecules  take  part  in  the  reaction.  The  velocity  constant 
ki  of  the  decomposition  of  the  substrate  is 


CHEMICAL  STATICS  IN  ENZYME  REACTIONS          253 

proportional  to  the  concentration  of  the  molecules  of  the  enzyme- 
substrate  compound;   or  if 

_  [enzyme-substrate] 

[enzyme]  [substrate]  ' 
then 

ki=Ki[enzyme]  [substrate]. 

In  a  similar  manner  the  velocity  constant  of  the  forma- 
tion   of    the    substrate    is  expressed  by 

£2  =  .^[enzyme]  [reaction  product]2, 

if,  as  is  often  the  case,  2  mols.  of  reaction  product  are  formed  from 
1  mol.  of  substrate. 

According  tovan't    Hoff,  the  equilibrium  is  then  given 
by  the  quotients 

KI  [substrate] 


_      _ 
~ 


^[reaction  product]2' 


As  will  be  at  once  seen,  the  numerical  value  of  this  "  enzymic  " 
end-state  coincides  with  that  of  the  natural  stable  equilibrium  as 
reached  with  an  inorganic  catalyst,  only  if  K\=K%,  hence  only 
in  the  case  where  the  combinations  enzyme-substrate  and  enzyme- 
reaction  product  are  exactly  equal.  No  convincing  cause  has, 
however,  yet  been  suggested  for  such  an  assumption;  on  the 
contrary,  the  results  obtained  by  Henri  with  invertase  bear 
the  interpretation  that  this  enzyme  is  combined  equally  by 
cane-sugar,  glucose  and  fructose. 

If  the  simple  assumption,  that  equal  numbers  of  the  two 
"  active  "  molecules  react  per  unit  of  time,  is  abandoned  and  it 
is  assumed  that  n%  of  enzyme-substrate  and  m%  of  enzyme- 
reaction  product  molecules  react  in  equal  times,  then 

_g  -  Jil  _         KI  ^[substrate] 

&2     J£2?w  [reaction  product]2' 

and  K  is  identical  with  the  constant  of  stable  equilibrium  if 


Hence  it  will  in  general  be  expected  that  enzymes  lead  to  an 
end-state  different  from  that  given  by  inorganic  catalysts  and 


254  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

the  first  question  to  be  decided  is  the  position  of  the  natural 
equilibrium.  The  natural  equilibrium  can  be  determined  directly 
in  many  cases,  for  instance,  in  the  system  fatty  acid-alcohol- 
ester-water.  In  order  to  accelerate  the  attainment  of  equilibrium, 
a  strong  mineral  acid  may  be  employed,  since  the  position  of 
equilibrium  is  not  altered  by  such  an  ideal  catalyst.  In  certain 
other  cases,  an  indication  of  the  position  of  equilibrium  may 
be  obtained  from  the  heat-change  of  the  reaction  (van't  Hoff, 
Sitzungsber.  K.  Akad.  Berlin,  1909,  42,  1065). 

As  was  shown  above,  the  heat-effects  of  enzymic  processes 
are  mostly  very  small.  For  such  changes,  however,  the  equi- 
librium is  of  a  simple  nature.  "  Optical  antipodes  which  form 
no  racemic  compound  present  the  ideal  example,  and  it  has 
been  shown  both  theoretically  and  experimentally  that,  in  the 
solid  state,  the  two  antipodes  are  in  equilibrium,  whilst  in  the 
vaporous,  fused  and  dissolved  conditions,  they  form  an  inactive 
mixture  of  equal  amounts.  The  relation  between  the  equilib- 
rium constant  K,  i.e.,  the  quotient  of  the  concentrations  of  the 
two  antipodes,  and  the  work  of  transformation  E  may  be  expressed 
thermodynamically  (van't  Hoff,  Svenska  Vet.  Akad. 
Handl.,  1886)  by 


Hence  in  this  case  #  =  0  and  K  —  l. 

What  applies  strictly  to  optical  antipodes  also  holds  approx- 
imately with  reactions  of  small  heat-effect  and  the  equilibrium 
is  not  far  removed  from  that  corresponding  with  thermo-neutrality. 

How  far  true  enzymic  equilibrium  may  differ  from  the 
natural  equilibrium  cannot  be  stated  exactly.  It  will  depend 
on  the  proportion  of  the  components  of  the  equilibrium  which 
combines  with  the  enzyme  or  —  if  it  is  assumed  that  separate 
enzymes  accelerate  the  reaction  in  the  two  directions  —  two 
enzymes  to  form  complex  compounds.  Since  the  concentration 
of  the  enzyme  is  usually  very  low,  the  concentrations  of  the  mole- 
cules of  enzyme-substrate  and  enzyme-reaction  product  must  also 
be  low,  and  the  enzymic  and  natural  equilibria  will  then  differ 
but  little.  In  other  words,  if  the  corresponding  enzyme 
or  mixture  of  enzymes  is  added  to  &  system  in  equilibrium,  the 


CHEMICAL  STATICS  IN  ENZYME  REACTIONS         255 

latter  undergoes  only  slight  change.  This  is  required  by  thermo- 
dynamics, which  also  shows  that  in  the  case  where  the  catalyst 
and  the  reacting  substances  do  not  (practically)  unite,  no  change 
in  the  equilibrium  should  be  produced.  Otherwise  the  equilib- 
rium could  be  altered  by  alternate  removal  and  introduction 
of  the  catalyst  and  perpetual  motion  thus  attained. 


B.    End-states    and    Stationary    States 

Starting  with  a  system  not  in  chemical  equilibrium,  the 
natural  equilibrium  is  not  necessarily  arrived  at  by  addition  of 
an  enzyme-preparation  capable  of  acting  on  the  system.  An 
end-state  differing  from  the  equilibrium  will  be  attained. 

1 .  If  two  enzymes  exist  which  catalyse  the  reaction  in  opposite 
directions.     The  final  state  then  depends  on  the  relative  quan- 
tities of  these  two  enzymes. 

2.  If  two  enzymes  are  present,  one  catalysing  the  formation 
of  a  compound  A  of  the  components  by  the  non-reversible  reaction 

Enzyme  1 

B+C    -*    A, 

and  the  other  using  up  this  compound  A  according  to  another 
r.on-reversible  reaction. 

Enzyme  2 

A    -»    D+F. 

This  case  may  evidently  lead  to  widely  varying  stationary 
states,  depending  on  the  relative  concentrations  of  the  two 
enzymes  1  and  2. 

T  a  m  m  a  n  n  (H.,  1892,  16,  271)  gave  an  account  of  the 
experimental  material  obtained  before  1892  and  also  of  his  own 
investigations.  From  the  results  of  the  latter  he  deduced  the 
important  and  undoubtedly  correct  consequence,  that  the  end- 
states  of  enzymic  reactions  do  not  coincide  with  the  positions  of 
stable  equilibrium  of  the  reactions. 

Concerning  the  end-states  attained  under  the  influence  of 
lipases,  two  more  recent  papers  have  been  published: 


256  GENEBAL  CHEMISTRY  OF  THE   ENZYMES 

Bodenstein  and  D  i  e  t  z  (Zeitschr.  f.  Elektrochem., 
1906,  12,  605)  have  compared  the  equilibrium  formed  between 
amyl  butyrate,  water,  amyl  alcohol  and  butyric  acid  with  the 
end-state  attained  by  this  system  under  the  influence  of  lipase. 
The  measurements  of  the  velocity-constants,  k\  and  £2,  with 
which  the  formation  and  resolution  of  the  ester  proceed,  have 
already  been  referred  to  on  p.  152.  The  mean  values  obtained 
were: 

ki  -0-015  &2  = 


As  should  theoretically  be  the  case,  the  quotient  of  these 
two  velocity  constants  was  found  to  be  equal  to  the  equilibrium 
constant  determined  directly.1 

Hence 


The  end-state  determined  in  these  two  ways  showed,  however, 
considerable  and  regular  deviations  from  the  natural  stable  equi- 
librium. Thus,  while  the  natural  equilibrium  constant  had  the 
value  1-96,  the  enzyme  experiments  gave  the  following  results: 

Initial  concentration  of  „ 

the  reacting  substances.  •& 

0-05  0-45 

0-10  0-74 

0-20  1-12 

The  fact  that  these  end-states  were  reached  from  both  direc- 
tions proved  that  they  were  not  dependent  on  retardations  of 
the  reaction. 

Unfortunately,  these  data  cannot  serve  for  proving  the  above 
relations  quantitatively,  since  the  system  examined  was  hetero- 
geneous (macro-heterogeneous) . 

A.  E.  Taylor  (Journ.  of  Biol.  Chem.,  1906,  2,  87)  also 
worked  with  Ricinus-lipase  in  the  form  of  a  moderately  finely- 
divided  suspension.  The  substrate  employed  was  triacetin, 
the  triglyceride  of  acetic  acid.  The  natural  equilibrium  was 
investigated  with  0-5,  1-0  and  2-0%  solutions  of  the  triacetin; 

1  Since  water  and  amyl  alcohol  were  present  in  excess,  the  constant  K 
simplifies  to 

[amyl  butyrate] 
[butyric  acid] 


CHEMICAL   STATICS  IN  ENZYME  REACTIONS          257 

mixtures  of  equal   volumes  of  these  solutions  and  of  normal 
sulphuric  acid,  left  for  several  months,  gave  the  values: 

Initial  concentration  of  the  ester.         Composition  of  the  equilibrated  liquid. 

0-5%  12%  ester,  88%  hydrolysed 

1  18       •"      82 

2  22         "      78 

For  the  enzymic  end-state,  the  following  numbers  were 
obtained : 

Initial  concentration  of  the  eater.  Composition  at  the  end-state. 

0-5%  14%  ester,  86%  hydrolysed 

1  21         "      79  " 

2  30        "      70 

From  these  numbers,  Taylor  drew  the  conclusion  that 
the  enzyme  does  not  displace  the  equilibrium;  but  the  dif- 
ferences between  these  two  series  of  numbers  are  so  large  and  so 
regular  that,  in  the  author's  opinion,  they  do  not  indicate  identity 
of  the  natural  and  enzymic  equilibria.  Whether  such  a  dif- 
ference exists  generally  and  how  it  depends  on  the  concentrations 
of  enzyme  and  substrate  are  questions  of  great  interest,  and  exper- 
iments in  this  direction  should  give  valuable  results. 

Scarcely  any  other  quantitative  determinations  of  enzymic 
end-states  have  been  made  which  are  comparable  with  the  natural 
equilibria.  From  Croft  Hill's  results  on  the  equilibrium 
between  maltose  and  glucose,  Pomeranz  (Wien.  Sitzungsber., 
II  B,  1902,  111,  554)  has,  indeed,  calculated  the  equilibrium  con- 
stants, which  are  in  good  agreement.  From  a  qualitative  point 
of  view  however,  this  end-state  is  by  no  means  clear;  maltose 
is  re-formed  either  not  at  all  or  only  in  inappreciable  amounts, 
being  replaced  by  dextrins  and  isomaltose. 

A  paper  communicated  byvan't  Hoff,  shortly  before 
his  death,  to  the  Berlin  Academy  of  Sciences  (Sitzungsber.  K. 
Akad.  Berlin,  1910,  48,  963),  treats  of  the  equilibrium  of  glucosides 
in  presence  of  emulsin. 

Measurements  were  made  first  with  the  natural  glucoside 
salicin,  in  presence  of  solid  salicin  and  solid  saligenin.  It  was 
found  that  the  formation  of  solid  salicin  from  the  solid  products 
of  hydrolysis  is  accompanied  by  an  expansion  in  volume  of 
9-47  c.c.  per  grm.-molecule.  The  result  was  that  the  hydrolysis 


258 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


of  salicin  proceeds  to  practical  completion;  the  equilibrium  was, 
however,  not  measurable.  [Also  V  i  s  s  e  r  (Zeitschr.  f.  physikal. 
Chem.,  1905,  52,  257)  had  previously  obtained  only  indications 
of  a  synthesis  of  salicin.] 

With  arbutin  and  aescillin,  the  hydrolyses  were  also  virtually 
complete. 

On  the  other  hand,  the  system  glycerol-glucose-water-glyc- 
erolglucoside  gives  a  measurable  condition  of  equilibrium. 
Experiments  were  made  with  the  molecular  proportion  1  :  4 
between  glucose  and  glycerol  and  with  increasing  amounts  of 
water.  If  the  number  of  mols.  of  the  latter  is  expressed  by  b 
and  the  fraction  of  the  glucose  changed  by  a,  then 


glucosideX  water         a  (6+ a) 


glucose  X  glycerol     ( 1  —  a)  (4 — a) 


=  k. 


Formation  of  glucose  still  occurred  with  6  =  15  and  a  =  0-38,  the 
value  for  k  being  2-6.  With  molecular  proportions  of  glycerol 
and  glucose,  as  much  as  70%  may  undergo  change. 

T  a  m  m  a  n  n  has  investigated  experimentally  the  dependence 
of  the  end-state  on  the  quantities  of  the  enzyme  and  of  the  react- 
ing compounds.  In  the  action  of  emulsin  on  arbutin  and  on 
coniferin,  it  was  found  that  the  amount  of  substance  hydrolysed 
at  the  end-state  increases  to  a  maximum  as  the  quantity  of 
enzyme  increases.  This  indicates  that,  with  increasing  concen- 
tration of  the  enzyme,  the  number  of  the  active  molecules, 
enzyme-product  of  reaction,  increases  more  than  that  of  the 
molecules  enzyme-substrate. 

When  constant  amounts  of  emulsin  and  different  amounts  of 
amygdalin  are  dissolved  in  25  c.c.  of  water,  the  following  amounts  of 
amygdalin  are  decomposed  at  40°: 


Original  quantity 
of  amygdalin. 

Amounts  hydrolysed 

Absolute  amounts. 

Percentage  amounts. 

0-51  grm. 
1-02  grms. 
2-04     " 

0-11  grm. 
0-15    " 
0-24    " 

20 
15 
12 

CHEMICAL  STATICS  IN  ENZYME  REACTIONS 


259 


Similar  relations  are  found  for  the  end-states  of  the  system  emulsin- 
arbutin.  The  solution  contained  0  •  0625  grm.  of  emulsin  and  the  follow- 
ing amounts  of  arbutin  at  35°: 


Original  quantity 
of  arbutin. 

Hydrolysed  after 

48  hours. 

72  hours. 

0-576  grm. 
4-000  grms. 

52-3% 
44-0 

52-3% 
44-0 

With  constant  amounts  of  enzyme,  relatively  more  amygdalin 
and  arbutin  are  hydrolysed  in  dilute  than  in  concentrated  solutions; 
the  same  probably  holds  for  the  hydrolysis  of  coniferin  by  emulsin. 

The  question  now  arises :  Within  what  limits  is  the  e  n  d  - 
state  of  an  enzymic  chemical  system  variable?  That  these  limits 
must  be  quite  wide  is  shown  at  once  by  the  above  facts.  They  depend 
not  only  on  the  concentration,  but  also  on  the  previous  history  of  the 
enzyme. 

Of  the  equilibria  of  biological  importance  with  which  considerable 
variations  have  been  observed,  that  between  starch  and  sugar  in  the 
living  plant  deserves  special  mention.  The  synthesis,  but  not 
the  hydrolysis  of  starch  is  largely  influenced  by  even  trifling  variations 
of  temperature  or  by  narcosis.  These  phenomena  may  be  explained  in 
two  ways: 

(1)  The  existence  may  be  assumed  of  two  different  enzymes,  one 
responsible  for  the  synthesis  and  the  other  for  the  hydrolysis;  this  is, 
of  course,  only  conceivable  on  the  supposition  that  different  quantities 
of  the  two  enzymes  combine  preferably  with  the  starch  or  sugar,  so 
that  both  starch  and  sugar  participate  in  two  equilibria: 


and 


[enzyme  a][starch]  =&m[enzyme  a— starch] 
[enzyme  b] [starch]  =/cw[enzyme  b—  starch]. 


If  corresponding  equilibria  hold  for  the  compounds  of  the  two 
enzymes  with  the  sugar,  it  is  obvious  that  there  arise  two  enzymic  end- 
states,  which  may  assume  widely  different  values.  Their  relations,  one 
to  the  other,  depend  only  on  the  total  concentration  of  the  starch.  None 
of  the  facts  are  contradictory  to  this  hypothesis,  for  which,  however,  no 
experimental  proof  is  forthcoming.  In  such  a  case,  indeed,  the  enzymes 
are  far  removed  from  ideal  catalysts.  (2)  Or  it  remains  to  be  tried 
— as  the  author  has  emphasised  in  another  place  (Pflanzenchemie,  II  and 
III,  p.  237) — whether  the  assumption  of  a  single  catalyst  or  a  single 


260  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

equilibrium  constant  does  not  meet  the  case.  The  very  varying  ways  in 
which  the  opposed  processes  of  building-up  and  breaking  down  react 
towards  external  influences,  would  then  be  attributed  to  the  different 
constitutions  of  the  media  in  which  condensation  and  hydrolysis 
proceed.  The  catalyst  and  the  reacting  substances  are  then  regarded  as 
distributed  between  the  aqueous  cell-sap  and  the  protoplasmic 
hydrosol.  In  the  former,  owing  to  the  great  excess  of  water  present,  the 
hydrolysis  may  proceed  far;  but  in  the  protoplasts,  which  are  rich  in 
lipoids  and  proteins,  such  a  relatively  small  proportion  of  water  dissolves 
that,  with  a  given  attainable  magnitude  of  the  sugar-concentration,  the 
opposite  reversionary  changes  predominate. 


CHAPTER  VI I 
ENZYMIC    SYNTHESES 

THE  suggestion  expressed  byvan't  Hoff  in  1898,  that 
enzymes  are  able  to  effect  or  accelerate  chemical  syntheses  (Zeitschr. 
f.  anorg.  Chem.,  1898,  18,  1),  has  since  then  been  confirmed  by 
the  results  of  numerous  investigations. 

It  was  in  the  above  year  that  Croft  Hill  (Journ.  Chem. 
Soc.,  1898,  73,  634)  observed  a  synthetic  action  in  the  case  of 
y  east-malt  ase. 

Croft  Hill  found  that  when  yeast-maltase  is  allowed 
to  act  for  a  month  at  30°  on  a  40%  solution  of  glucose,  the 
reducing  and  rotatory  powers  of  the  solution  are  so  altered  as  to 
indicate  formation  of  maltose.  Shortly  afterwards,  however, 
E  m  m  e  r  1  i  n  g  (Chem.  Ber.,  1901,  34,  600,  2207)  showed  that 
the  effect  observed  by  C  r  o  f  t  Hill  depends  on  the  formation, 
not  of  maltose,  but  of  isomaltose  and  dextrinous  products. 
Isomaltose  is  not  again  hydrolysed  by  maltase.  Similar  behaviour 
was  noted  byE.  Fischer  and  E.  F.  Armstrong  (Chem. 
Ber.,  1902,  35,  3144)  with  kephir-lactase,  which  from  galactose 
and  glucose  synthesises  not  lactose  but  isolactose,  a  carbohydrate 
not  attacked  by  the  lactase.  Finally,  E.F.  Armstrong 
(Proc.  Roy.  Soc.,  B,  1904,  73,  516)  made  a  number  of  interesting 
observations  which  extended  the  discovery  of  E  m  m  e  r  1  i  n  g 
referred  to  above:  the  behaviour  of  emulsin  is  the  opposite 
of  that  of  maltase,  as  it  hydrolyses  isomaltose  but  synthesises 
glucose  to  maltose.  These  results  led  Armstrong  to  the 
generalisation  that  "  Enzymes  build  up  just  those  molecules 
which  they  are  unable  to  break  down." 

This  is  a  problem  of  fundamental  theoretical  importance, 
If  Armstrong's  view  is  correct,  it  must  be  assumed  thai 
those  enzymes  which  give  rise  to  a  chemical  equilibrium  froir 
both  directions  represent  mixtures  of  a  synthesising  and  a  hydro- 
lysing  enzyme.  Against  the  admissibility  of  this  hypothesis 

261 


262  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

which  is  only  weakly  supported  by  experiment,  no  fundamental 
objection  can  be  advanced,  and  we  may  again  consider  the  facts 
favouring  such  a  two-enzyme  theory.  It  has  been  emphasised, 
especially  by  Bayliss,  that  the  experimental  foundation 
for  this  view  is  indeed  rather  weak,  inasmuch  as  Croft  Hill 
made  use  of  ordinary  brewers'  yeast,  which  has  been  shown 
by  H  e  n  r  y  and  A  u  1  d  (Proc.  Roy.  Soc.,  B,  1905,  76,  568) 
to  contain  "  emulsin  "  and  hence  a  ^-glucosidase.  This  emulsin 
may  have  occasioned  the  formation  of  isomaltose.  The  forma- 
tion of  isomaltose  and  isolactose  admits,  however,  of  another 
possible  interpretation,  which  has  been  given  by  E .  F .  Arm- 
strong (Proc.  Roy.  Soc.,  B, 1905,  76>  513). 

It  has  been  known  since  O  '  S  u  1 1  i  v  a  n  and  Tompson's 
work,  and  has  been  confirmed  by  H  u  d  s  o  n  ,  that  the  hydrolysis 
of  cane-sugar  yields  a  d-glucose  distinguished  by  its  high  rotatory 
power;  this  sugar,  to  which  T  a  n  r  e  t — to  whom  we  owe  a  very 
complete  investigation  of  this  sugar — gave  the  name  a-glucose, 
passes  gradually  into  e-glucose.  This  s-glucose  appears  to  con- 
sist of  a-  and  ^-glucoses  in  equilibrium.  In  aqueous  solution  there 
is  little  a-glucose  and  a  relatively  large  proportion  of  ^-glucose. 
On  adding  to  such  a  solution,  an  enzyme  the  synthetic  action  of 
which  is  to  be  studied,  an  excess  of  the  ^-modification  is  available 
and  it  is  to  be  expected  that  the  synthetic  biose  will  contain  the 
glucose-residue  mainly  in  a  form  corresponding  with  ^-glucose. 
The  biose  corresponding  with  the  oc-hexose  should  also  be  formed 
in  smaller  amount  at  the  same  time,  but  this  has  not  been  observed. 
Nothing,  however,  is  yet  known  as  to  what  constitutes  the  dif- 
ferences between  these  modifications. 

This  view  is  capable  of  experimental  proof  in  various  ways. 

Firstly,  it  might  be  expected  that  the  bioses,  such  as  iso- 
maltose, synthesised  from  glucose  solutions,  should  yield  ^-glucose 
directly,  but  no  such  result  appears  to  have  been  obtained; 
this  should  also  be  the  case  with  those  bioses  and  glucosides  which 
undergo  the  same  enzymic  hydrolyses.  Further,  it  would  be 
expected  that  maltose  could  be  synthesised  from  a-glucose — • 
i.e.,  from  a  freshly-prepared  solution  of  glucose — and  a  very 
active  enzyme  capable  of  effecting  the  synthesis,  before  the  change 
from  a-  to  ^-modification  is  complete.1 

1  With  reference  to  certain  statements  in  the  literature,  it  must  be  pointed 
out  that  originally,  the  names  a-glucose  and  a-glucoside  were  not  connected. 


ENZYMIC  SYNTHESES 


263 


Apart  from  the  facts  mentioned  above  with  reference  to  the 
synthesis  of  maltose  and  lactose,  numerous  other  statements 
have  been  made  relating  to  enzymic  syntheses  which  are  regarded 
as  pure  reversions. 

Ethyl  butyrate  is  formed  from  butyric  acid  and  ethyl  alcohol 
by  the  action  of  pancreas-lipase  (K  a  s  1 1  e  and  L  o  e  v  e  n  - 
hart,  Amer.  Chem.  Journ.,  1900,  24,  491). 

Glyceryl  butyrate  (H  a  n  r  i  o  t ,  C.  R.,  1901,  132,  212)  and 
amyl  butyrate  (Bodenstein  and  D  i  e  t  z  ,  Zeitschr.  f . 
Elektrochem.,  1906,  12,  605)  are  also  formed  from  their  com- 
ponents. 

An  extensive  series  of  experiments  on  the  formation  of  ester 
from  methyl  alcohol  and  oleic  acid  by  pancreas-lipase  has  been 
carried  out  by  Pottevin  (Bull.  Soc.  Chim.,  1906,  35,  693; 
Ann.  Inst.  Pasteur,  1906,  20,  901).  These  show,  among  other 
results,  that  the  equilibrium  between  glycerol  and  oleic  acid  is 
independent  of  the  quantity  of  enzyme  added. 


Quantity  of 

Percentage  of  ester  formed. 

pancreatin  employed. 

1  day. 

2  days. 

20  days. 

1 

8 

56 

84 

2 

12 

66 

82 

5 

21 

66 

84 

10 

43 

74 

85 

The  following  results,   relating  to  the  synthesis   of  a  true 
fat,  indicate  the  influence  of  the  amount  of  water  present. 


40  grms.  of  oleic   acid    +  3  grms.   powdered   pancreas, 
experiment,  20  days.     Temperature,  33°. 


Duration  of 


Amounts  of 

Percentage  esterified. 

Glycerol. 

Water. 

130  grms. 

0  grms. 

3 

120 

10 

77 

110 

20 

64 

100 

30 

51 

64 

66 

20 

28 

102 

5 

8 

122 

0 

264  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Syntheses  of  true  fats  from  various  higher  fatty  acids  (of 
(ocoanut  oil,  etc.)  and  glycerol  are  described  by  Welter 
cZeitschr.  f.  angew.  Chem.,  1911,  24,  385). 

Glyceryltriacetate  is  formed  from  its  components  by  Ricinus- 
lipase  (Taylor,  Journ.  of  Biol.  Chem.,  1906,  2,  87).  Cf. 
p.  154. 

Amygdalin  is  formed  from  mandelonitrile  glucoside  and 
glucose  (E  m  m  e  r  1  i  n  g  ,  Chem.  Ber.,  1901,  34,  3810). 

Benzaldehydecyanohydrin  results  from  benzaldehyde  and 
hydrocyanic  acid  (Rosenthaler,  Biochem.  Z.,  1908,  14, 
238;  1909,  19,  186). 

Triacetylglucose  is  given  by  acetic  acid  and  glucose  under 
the  influence  of  pancreatin  (A  c  r  e  e  and  H  i  n  k  i  n  s  ,  Amer. 
Chem.  Journ.,  1902,  28,  370). 

Glycogen  is  formed  in  pressed  yeast-juice  from  sugar 
(C  r  e  m  e  r  ,  Chem.  Ber.,  1899,  32,  2062). 

A  condensation,  the  nature  of  which  is  not  clearly  understood, 
is  produced  in  invert-sugar  solutions  by  the  revertase  of 
Mucor  mucedo,  etc.  (P  a  ri  t  a  n  e  1 1  i ,  Atti  Real.  Accad. 
Lincei,  1907,  [v],  16,  ii,  419;  Bot.  Ber.,  1908,  26a,  494).  Also 
no  definite  conclusions  can  be  drawn  concerning  the  action  of 
the  yeast-revertase  investigated  by  Kohl  (Beitr.  z.  bot.  Zen- 
tralbl.,  1908,  23,  i,  64). 

Reference  must  also  be  made  here  to  the  studies  of 
Maquenne  (Bull.  Soc.  Chim.,  1906,  [iii],  35;  lecture)  and  of 
Wolff  and  Fern  bach  (C.  R.,  1903,  137,  718)  on  the 
re-formation  of  amylose  from  its  decomposition  products;  men- 
tion should  also  be  made  ojf  the  equilibrium  attained  under  the 
action  of  malt-diastase  (M  o  r  i  t  z  and  Glendinning, 
Journ.  Chem.  Soc.,  1892,  61,  689).  Possibly  the  reversion  of 
starch  in  plasmolysed  vegetable  cells,  observed  by  Overton 
(Vierteljahrsschr.  d.  naturf.  Ges.  in  Zurich,  1899,  44,  88),  also 
represents  such  an  enzyme  action. 

Kendall  and  Sherman  (Journ.  Amer.  Chem.  Soc., 
1910,  32,  1087)  found  that  a  state  of  equilibrium  is  also  set  up  in 
the  decomposition  of  starch  by  amylase  (pancreas-amylase) .  In 
1  per  cent  starch  solution,  equilibrium  is  attained — independently 
of  the  amounts  of  salt  and  alkali  present — when  the  amount  of 
maltose  is  about  85%  of  the  initial  weight  of  the  starch. 

The  formation  of  hippuric  acid  from  benzoic  acid   (benzyl 


ENZYMIC  SYNTHESES  265 

alcohol)  and  glycine  by  the  action  of  kidney-extract  has  been 
observed  by  Abelous  and  Ribaut  (Soc.  Biol.,  1900, 
52,  543),  but  confirmation  of  this  result  is  desirable. 

As  regards  the  synthesis  of  protein  substances,  mention 
must  first  be  made  of  the  experiments  on  plastein-formation, 
which  must  undoubtedly  be  regarded  as  syntheses. 

D  a  n  i  1  e  w  s  k  i  established  the  fact  that,  in  concentrated 
solutions  of  Witte's  peptone,  rennet  produces  characteristic 
protein  precipitates.  This  phenomenon,  "  plastein-formation," 
which  also  occurs  under  the  influence  of  pepsin  preparations, 
was  further  investigated  in  Danilewski's  laboratory  and 
has  since  been  examined  more  especially  by  Russian  workers, 
for  instance,  S  aw  j  alow  (Centralbl.  f.  Physiol.,  1902,  16, 
625)  and  Okuneff  (Dissertation,  St.  Petersburg,  1895). 
Kurajeff  (Hofm.  Beitr.,  1901,  1,  121;  1903,  4,  476)  found 
papain  to  possess  a  similar  coagulating  property.  L  a  w  r  o  w 
and  S  a  1  a  s  k  i  n  (H.,  1902,  36,  277)  showed  that  the  precipita- 
tion of  concentrated  Witte's  peptone  solutions  by  gastric  juice 
occurs  with  albumoses  of  all  types.  Our  knowledge  of  plastein- 
formation  has  recently  (H.,  1907,  51,  1)  been  considerably 
extended  by  L  a  w  r  o  w ,  according  to  whom,  not  only 
the  albumoses  but  also  substances  of  the  amino-acid  type 
can  be  coagulated  best  in  faintly  alkaline  solution.  The 
coagulums  exhibit  the  reactions  of  proteins  but  contain  less 
nitrogen  than  these. 

Plastein-formation  is  favoured  by  increasing  the  concen- 
trations of  the  reacting  solutions  and  occurs  especially  under 
conditions  which  retard  the  hydrolysis  of  proteins. 

The  precipitation  of  plasteins  may  possibly  consist  of  a  salting- 
out  process. 

Everything  seems  to  indicate  that  Danilewski's  reac- 
tion is  really  a  synthesising  action  1  of  the  pepsin  or  rennet, 
although  true  reversibility  of  the  peptic  action,  i.e.-,  re-forma- 
tion of  the  starting  material  has  not  been  proved.  Rosen- 
f  e  1  d  (Hofm.  Beitr.,  1906,  9,  215)  has  shown  that  the  hydrolytic 
products  of  casein-plastein  differ,  at  any  rate  quantitatively, 
from  those  of  casein.  This  is  proved  very  clearly  by  a  more 
recent  investigation  of  Henriques  and  G  j  a  1  d  b  a  k  (H., 

1  Cf .  also  R.  O.  Herzog  (H.,  1903,  39,  305)  and  A  .  Number  g 
(Hofm.  Beitr.,  1903,  4,  543). 


266  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

1911,  71,  485),  who  followed  the  reaction  by  means  of  S  6  r  e  n  - 
s  e  n  's  method  of  titration  with  formaldehyde. 

An  interesting  pepsin-synthesis  from  the  hydrolytic  products 
of  casein  has  been  communicated  by  Robertson  (Journ. 
of  Biol.  Chem.,  1907,  3,  95)  who  obtained  a  body  which  con- 
tained 3-17%  P2Os  and  which  he  termed  u  paranuclein." 

Taylor  succeeded  in  effecting  an  enzymic  synthesis  with 
trypsin.  He  hydrolysed  protamine  sulphate  from  R  o  c  c  u  s 
1  i  n  a  t  u  s  completely  into  its  components  and  treated  the  mix- 
ture of  amino-acids  with  trypsin  obtained  from  the  liver  of  the 
mollusc  Schizothaerus  Nuttalii  (Journ.  of  Biol. 
Chem.,  1907,3,87). 

The  same  author  has  recently  described  further  experiments 
both  with  glycerine  liver-extract  of  Schizothaerus  Nut- 
talii and  with  pancreatin  (G  r  ii  b  1  e  r  '  s)  ,  the  substrate 
being  obtained  as  before  from  salmin  sulphate.  After  a  lapse  of 
4  months,  the  solutions,  which  proved  to  be  still  germ-free,  were 
diluted  with  4  parts  of  water,  acidified  with  sulphuric  acid  and 
mixed  with  3  parts  of  absolute  alcohol;  the  control  solutions 
showed  no  change,  whilst  the  two  containing  enzyme  gave  thick 
white  precipitates.  The  product  was  purified  by  means  of  its 
picrate,  after  which  it  was  found  to  have  a  composition  closely 
agreeing  with  that  of  salmin. 

Indications  of  a  synthetic  action  of  trypsin  on  the  hydro- 
lytic products  of  casein  were  obtained  by  B  a  y  1  i  s  s  (Arch. 
Sci.  Biol.  St.  Petersburg,  1904,  11,  Supplement,  261),  while  the 
number  of  cases  in  which  retardation  of  the  hydrolysis  by  the 
reaction  products  has-  been  observed  show  that  a  state  of  rever- 
sible equilibrium  is  assumed. 

The  experiments  of  Beitzke  and  N  e  u  b  e  r  g  (Verh. 
d.  deutsch.  path.  Ges.,  1905,  160;  Virch.  Arch.,  1906,  183,  169) 
are  of  special  interest,  both  in  themselves  and  in  relation  to  the 
representation  of  enzymic  end-states  given  on  p.  253  et  seq. 
It  was  found  that  subcutaneous  injection  of  emulsin  (in  rabbits) 
leads  to  the  formation  of  anti-emulsin,  which  is  able  to  synthesise 
glucose  to  maltose  or  to  a  disaccharide  similar  to  maltose. 

Should  further  experiments  show  that  our  enzyme-prepara- 
tions contain  in  general  a  resolving  and  a  synthesising  con- 
stituent, the  prevailing  views  concerning  the  formation  and  com- 
bination of  anti-enzymes  may  require  modification,  the  deter- 


ENZYMIC   SYNTHESES  267 

mining  factor  in  the  equilibrium  between  enzyme  and  anti- 
enzyme  being  ascribed  to  the  chemical  substrate  and  the  products 
it  yields. 

Anti-enzymes 

Like  the  toxines,  many  enzymes  are  able  to  cause  production, 
in  the  living  organism,  of  anti-bodies  which  retard  the  actions  of 
the  enzymes.  Anti-enzyme  action  was  first  observed  by 
Hildebrandt  in  1893. 

From  their  actions,  anti-enzymes  appear,  like  enzymes,  to  be 
organic  catalysts.  They  seem  to  correspond  with  the  enzymes 
in  physical  properties  and  also  in  chemical  lability,  although  the 
observations  in  this  direction  are  few  and  not  very  definite. 
They  are,  as  far  as  is  known,  approximately  as  unstable  towards 
high  temperatures  as  the  enzymes  themselves.  Among  the 
most  stable  anti-enzymes  are  the  anti-lipase  of  Ricinus  which, 
according  to  Bertarelli  (Centralbl.  f.  Bakt.,  1905,  I,  40, 
231),  is  not  weakened  at  70°  and  slowly  loses  its  activity  only  at 
80°.  The  degree  of  purity  of  the  enzyme  appears  to  be  without 
influence  on  the  formation  of  anti-body,  and  a  co-enzyme  is 
apparently  unnecessary  to  this  reaction. 

This  behaviour  corresponds  closely  with  that  of  the  antitoxines, 
which  are  generally  injured  at  70°.  Like  enzymes,  antitoxines  are  more 
stable  in  the  dry  state  than  in  solution. 

Judging  from  the  results  of  physico-chemical  investigations, 
the  relations  between  enzymes  and  anti-enzymes  are  essentially 
identical  with  those  between  toxines  and  anti-toxines.  For 
further  information  on  this  interesting  problem,  the  monographs 
of  Arrhenius  and  of  M  i  c  h  a  e  1  i  s  should  be  consulted. 
The  most  important  facts  concerning  anti-enzymes  are  here 
brought  together,  because  the  problem  of  the  reversibility  of 
enzymic  reactions  is  bound  up  with  the  action  of  anti-enzymes. 

By  the  term  anti-enzymes,  in  its  stricter  meaning,  is  to  be 
understood  those  specifically-acting  secretions  produced  in  the 
organism,  in  presence  of  enzymes,  by  immunisation.  But  normal 
serum  also  contains  substances  which,  for  instance,  annul  tryptic 
action  more  or  less  completely.  It  is  not  probable,  from  the  facts 
yet  known,  that  these  differ  essentially  from  the  anti-bodies 


268  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

produced  by  immunisation,  and  they  will  therefore  be  discussed 
with  the  anti-enzymes.  On  the  other  hand,  the  thermo-stable 
and  inorganic  substances  which  prevent  enzyme  action  to  a  greater 
or  less  extent  will  be  termed,  after  Neuberg's  suggestion, 
inhibiting  agents. 

Anti-steapsin  was  prepared  by  A.  Schlitze  (Deut.  med. 
Wochens.,  1904,  30,  308),  who,  after  injecting  steapsin,  obtained 
in  two  cases  rabbit-serum  showing  strong  anti-lipolytic  action. 
His  results  have  more  recently  been  confirmed  by  B  e  i  t  z  k  e 
and  N  e  u  b  e  r  g  (Virch.  Arch.,  1906,  183,  169)  and  by 
B  e  r  t  a  r  e  1 1  i  . 

The  latter  author  (Centralbl.  f.  Bakt.,  1905,  I,  40,  231)  was 
not  able  to  obtain  the  anti-enzymes  corresponding  with  the 
lipases  from  ox-liver  and  ox-blood  serum,  but  by  injection  of 
various  vegetable  lipases  (Ricinus-  and  nut-lipase)  he  separated 
from  dog-serum  anti-lipases  with  specific  actions.  Thus  the 
anti-ricinus-lipase  influenced  neither  the  serum-lipase,  nor  the 
liver-lipase,  nor  the  nut-lipase. 

Anti-emulsin,  the  first  known  anti-enzyme,  was 
discovered  by  Hildebrandt  (Virch.  Arch.,  1893,  131,  12) 
who,  like  B  e  i  t  z  k  e  and  N  e  u  b  e  r  g  (Virch.  Arch.,  1906, 
183,  169),  immunised  rabbits  and  precipitated  the  anti-body 
from  the  anti-ferment-serum  with  the  globulin  fraction.  The 
synthetic  action  of  this  preparation  is  discussed  later. 

Immunisation  with  diastase  may  perhaps  have  been  observed 
by  Kussmaul  (Arch.  f.  klin.  Med.,  14),  but  his  results  *are 
uncertain.  A  s  c  o  1  i '  s  work  (H.,  1904,  43,  156)  also  led  to 
no  definite  result. 

Subcutaneous  injection  of  diamalt — a  commercial  enzyme 
solution  prepared  from  green  malt — leads  to  the  production  in 
rabbit-serum  of  substances  which  retard  the  saccharifying  action 
of  diastase,  while  the  serum  originally  showed  an  inverting  action 
(B  r  a  u  n  and  S  c  h  ii  t  z  e  ,  Med.  Klin.,  1907,  No.  9,  quoted 
in  Biochem.  Zentralbl.,  1907,  6,  389). 

S  a  i  k  i  prepared  an  anti-inulinase  by  injection 
(Journ.  of  Biol.  Chem.,  1997,  3,  395). 

An  anti-invertase  of  slight  activity  was  obtained  by 
Schtitze  and  Berg  ell  (Zeitschr.  klin.  Med.,  1907,  61,  366). 
S  c  h  ii  t  z  e  (Zeitschr.  f.  Hygiene,  1904,  48,  457)  also  prepared  an 
anti-lactase  by  injecting '  kephir-lactase  under  the  skin 


ENZYMIC  SYNTHESES  269 

of  rabbits  or  into  the  breast-muscles  of  the  dog;  the  anti-body 
appears  in  the  serum,  which  in  the  normal  state  did  not  contain  it. 

Anti-pepsin.  Sachs  ^(Fortschr.  d.  Med.,  1902,  20, 
425)  immunised  geese  against  pepsin,  the  serum  exhibiting 
sufficient  anti-peptic  action  to  annul  20  times  its  amount  of  pepsin. 

The  anti-pepsin  which  was  discovered  by  Weinland 
(Zeitschr.  f.  Biol.,  1903,  44,  45)  and  is  regarded  as  a  normal 
secretion  of  the  gastric  mucous  membrane,  corresponds  with  the 
normal  anti-trypsin  of  blood-serum.  It  retards  peptic  digestion 
in  vitro  and  doubtless  prevents  auto-digestion  of  the  mucous 
membranes. 

The  same  author  has  detected  anti-enzymes  of  pepsin  and 
trypsin  in  the  pressed  juice  of  Ascaris,  and,  according  to 
R.O.  Herzog  (H.,  1909,  60,  306),  the  action  of  rennet 
preparations  is  also  retarded  by  Ascaris  juice. 

Unlike  these  anti-enzymes,  an  agent  which  inhibits  peptic 
action  and  was  found  by  B  1  u  m  in  gastric  juice  is  stable  to  heat. 

While  B  e  r  g  e  1 1  and  S  c  h  ti  t  z  e  tried  in  vain  to  obtain  an 
anti-pancreatin  (Zeitschr.  f.  Hygiene,  1905,  50,  305),  J  o  c  h  - 
m  a  n  n  and  Kantorowicz,  in  a  recent  preliminary  com- 
munication (Munch,  med.  Wochens.,  1908,  55,  728),  refer  to 
an  anti-enzyme  to  pancreas-trypsin  which  must  be  identical 
with  the  anti-body  of  the  leucocyte-enzyme.  The  same  inves- 
tigators state  that  blood  contains  at  least  two  anti-pepsins,  one 
of  which  inhibits  the  digestion  of  serum-albumin,  being  destroyed 
at  80-85°,  while  the  other  prevents  the  digestion  of  solidified 
hens'  egg-albumin,  being  thermo-stable. 

Attempts  at  immunisation  against  papain  have  as  yet  been 
unsuccessful  (B  e  r  g  e  1 1  and  Schiitze,  loc.  cit.;  von 
Stenitzer,  Biochem.  Z.,  1908,  9,  382). 

Anti-tryptic    Paralysors 

The  experiments  of  H  a  h  n  (Berl.  klin.  Wochens.,  1897, 
34,  499)  and  those  made  almost  simultaneously  by  P  u  g  1  i  e  s  e 
and  Coggi  (Boll.  Sci.  Med.,  1897,  8)  first  established  the 
fact  that  normal  serum  retards  tryptic  digestion.  Fermi 
had,  however,  previously  observed  that  trypsin  disappears  soon 
after  injection.  Further  work  on  this  subject  has  been  done  by 
A  c  h  a  1  m  e  (Ann.  Inst.  Pasteur,  1901,  15,  737),  Camus  and 


270  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

G  1  e  y  (Soc.  Biol.,  1897,  47,  425),  Charrin  and  Levaditi, 
S  i  m  n  i  t  z  k  i  and  Glaessner  (Hofm.  Beitr.,  1903,  4, 
79).  This  anti-tryptic  action  is  bound  up  with  the  serum- 
albumin  (Landsteiner,  Centralbl.  f.  Bakt.,  1900,  I,  27, 
357;  Cathcart,  Journ.  of  Physiol.,  1904,  31,  497;  H  e  d  i  n  , 
Journ.  of  Physiol.,  1905,  32,  390).  With  a  number  of  diseases, 
such  as  diabetes,  tuberculosis,  etc.,  increased  anti-tryptic  activity 
of  the  serum  is  observed  (B  r  i  e  g  e  r  and  T  r  e  b  i  n  g  , 
Berl.  klin.  Wochens.,  1908,  45,  1041). 

According  to  A.  D  6  b  1  i  n  (Zeitschr.  f .  Immunitatsforsch. 
u.  exp.  Therap.,  1910,  4,  229),  the  anti-trypsin  of  serum  is  stable 
to  heat,  which  weakens  the  anti-tryptic  action  of  urine  only 
slightly.  The  inhibiting  body  is  not  a  lipoid  but  is  colloidal  in 
character. 

Delezenne  (Soc.  BioL,  1903,  55,  112)  found  that  the 
retarding  action  of  normal  serum  is  not  a  direct  action  on  the 
proteolytic  enzyme,  but  is  to  be  attributed  to  neutralisation  of 
the  corresponding  kinase.  He  describes  the  following  experiments: 

After  preliminary  determination  of  the  amount  of  serum  just 
necessary  to  annul  the  digestive  action  of  a  mixture  of  pancreatic 
and  intestinal  juices,  three  tubes  were  each  filled  with  equal 
quantities  of  the  substance  to  be  digested,  the  pancreatic-intestinal 
mixture  and  the  corresponding  quantity  of  serum.  After  the 
lapse  of  some  hours,  when  it  was  found  that  no  digestion  had 
occurred  in  any  of  the  tubes,  to  one  (A)  was  added  an  excess  of 
pancreatic  juice  and  to  another  (B)  an  excess  of  gastric  juice, 
while  C  served  as  control;  digestion  took  place  only  in  B.  From 
these  results  Delezenne  inferred  that,  in  the  digestion  with 
the  pancreatic-intestinal  juice  only  the  intestinal  juice  (the 
kinase)  was  neutralised,  and  that  the  serum  has  no  action  on  the 
pancreatic  enzyme.  A  s  c  o  1  i  and  B  e  z  z  o  1  a  (Centralbl.  f. 
Bakt.,  1903,  I,  33,  783)  arrived  at  somewhat  similar  conclusions. 

Against  this  view  objections  have,  however,  been  advanced. 
According  to  Delezenne,  the  anti-trypsin  would  be  an  anti- 
kinase,  and.  the  existence  of  an  anti-trypsinogen  might  also  be 
expected.  B  a  y  1  i  s  s  and  Starling  (Journ.  of  Physiol., 
1904,  32,  129)  were  unable  to  detect  such  a  body  in  blood-serum 
after  subcutaneous  injection.  It  was  also  shown  that  normal 
rabbit-serum  possesses,  in  addition  to  its  anti-tryptic  properties, 
the  ability  to  neutralise  enterokinase,  this  power  being  enhanced 


ENZYMIC   SYNTHESES  271 

by  injection  of  enterokinase.  On  the  other  hand,  "  anti-kinase  " 
produced  in  serum  does  not  increase  its  anti-tryptic  properties. 

These  observations  have  been  confirmed  and  extended  by 
Zunz  (Bull.  Acad.  Roy.  Med.  de  Belgique,  1905,  [4],  19). 

Normal  blood-serum  contains  not  only  anti-trypsin,  but 
also  a  proteolytic  enzyme,  serum-protease,  the  action  of  which 
is  retarded  by  the  anti-trypsin.  Serum-protease  can  be  separated 
from  anti-trypsin  by  salting-out,  the  former  passing  into  the 
globulin  fraction  and  the  latter  into  the  albumin  fraction. 

That  the  amount  of  anti-trypsin  in  serum  can  be  increased 
considerably  by  injection  of  trypsin  solutions,  has  been  shown 
by  Achalme  (Ann.  Inst.  Pasteur,  1901,  15,  737)  and  by 
W  e  i  n  1  a  n  d  (Zeitschr.  f.  Biol.,  1902,  44,  1,  45). 

As  regards  the  sensitiveness  of  the  "  anti-proteolase "  to 
heat,  Vandevelde's  experiments  (Biochem.  Z.,  1909,  18, 
142)  appear  to  indicate  that  weakening  takes  place  at  55°. 

According  to  F  e  r  m  i  (Centralbl.  f.  Bakt.,  1909,  I,  50,  225), 
anti-tryptic  action  is  exhibited  by  various  organic  tissues  and 
by  certain  protein  substances,  such  as  yolk  of  egg  and  milk; 
casein  alone  also  has  an  anti-tryptic  action. 

The  clinical  significance  of  the  tryptases  and  anti-tryptases, 
which  cannot  be  treated  in  detail  here,  will  be  found  discussed 
in  a  comprehensive  paper  by  von  Bergmann  and  Kurt 
Meyer  (Berl.  klin.  Wochens.,  1908,  45,  1673). 

Anti-urease.  As  was  discovered  by  L  .  Moll  (Hofm. 
Beitr.,  1902,  2,  344),  normal  (rabbit-)  serum  and  normal  albumin- 
free  urine  always  exert  a  retarding  action  on  urease.  This  action 
is  markedly  increased  by  injection  of  small  doses  of  a  urease- 
preparation  from  Micrococcus  ureae  Pasteuri. 
Moll  does  not  regard  the  anti-bodies  of  normal  and  of  immu- 
nised serum  as  identical,  since  the  latter  loses  the  excess  of  its 
inhibiting  power  and  hence  becomes  normal  in  this  respect  if 
heated  for  an  hour  at  65°  (but  not  at  56°),  whilst  the  retarding 
capacity  of  normal  serum  is  not  altered  by  heating  either  for  an 
hour  at  65°  or  for  six  hours  at  56°. 

Anti-bodies  of  clotting  enzymes.  The 
important  discovery  of  Morgenroth,  that  subcutaneous 
injection  of  rennet  produces  an  anti-rennet  in  the  serum 
and  milk  of  the  immunised  animal  (Centralbl.  f.  Bakt.,  1899, 
26,  349;  1900,  27,  721),  directed  attention  to  these  anti-enzymes. 


272  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

As  regards  anti-rennet  itself,  Morgenroth  and  more  especially 
M  a  d  s  e  n  and  W  a  1  b  u  m  ,  and  Bashford  have  inves- 
tigated its  action  quantitatively,  whilst  F  u  1  d  and  S  p  i  r  o  (H., 
1900,  31,  132)  have  made  a  comprehensive  study  of  the  rennetic 
and  anti-rennetic  action  of  the  blood.  Arrhenius  has 
pointed  out  the  analogy  existing  between  the  behaviour  of  the 
clotting  enzymes  and  that  of  certain  precipitins  towards  the 
anti-bodies.  It  is  hence  unnecessary  to  describe  the  equilibrium 
phenomena  between  rennet  or  the  fibrin-ferment  and  the  anti- 
bodies. 

Emphasis  must  be  laid  on  the  fact  discovered  by  H  a  m  - 
m  a  r  s  t  e  n  and  R  6  d  e  n  as  early  as  1887  that  the  normal 
serum  of  various  animals  contains  a  substance  which  inhibits  the 
action  of  rennet.  This  substance  is,  however,  not  identical  with 
that  produced  by  active  immunisation  (Bashford,  Journ. 
of  Pathol.,  1902,  8,52).  According  to  Fuld  and  Spiro, 
the  "  anti-rennet  "  contained  in  horse-blood  serum  is  a  pseudo- 
globulin  which  acts  by  fixing  a  portion  of  the  calcium  ions  and 
so  retarding  coagulation;  these  authors  separate  the  chymosin 
and  anti-chymosin  of  normal  blood  by  precipitation  with  ammo- 
nium sulphate. 

For  the  relations  between  anti-pepsin  and  anti-rennet,  see 
J  a  c  o  b  y  (Biochem.  Z.,  1907,  4,  471). 

Anti-fibrin-ferment.  Bordet  and  G  e  n  g  o  u 
(Ann.  Inst.  Pasteur,  1901,  15,  129)  obtained  this  anti-body  in 
the  following  manner:  They  injected  guinea-pigs  with  normal 
rabbit-serum,  by  which  means  the  guinea-pig-serum  acquires 
the  property  of  retarding  the  coagulation  of  rabbit-blood,  i.e., 
the  anti-fibrin-ferment  is  formed.  This  acts,  as  these  authors 
showed,  in  a  somewhat  markedly  specific  manner  on  the  sera 
of  different  animals.  This  anti-body  is  not  affected  by  heating 
to  55°. 

The  composition  of  the  anti-fibrin-ferment  is  not  definitely 
known. 

Anti-laccase  was  thought  to  have  been  obtained  by 
G  e  s  s  a  r  d  (Soc.  Biol.,  1903,  55,  227)  in  rabbit-serum  by  injec- 
tion of  laccase;  the  shortness  of  his  communication  renders 
criticism  of  the  work  impossible. 

The  anti-catalase  of  Battelli  and  Stern  (Soc. 
Biol.,  1905,  58,  235,  758)  behaves  like  ferric  sulphate  and  should  be 


ENZYMIC  SYNTHESES  273 

classed  with  the  inhibiting  agents.  A  true  anti-enzyme  of  catalase 
does  not  appear  to  exist  (D  e  W  a  e  1  e  and  Vandevelde, 
Biochem.  Z.,  1908,  9,  264). 

Although  anti-enzymes  corresponding  with  vegetable  enzymes 
have  often  been  produced  in  the  animal  body  (M  a  g  n  u  s  and 
Friedenthal),  no  appreciable  formation  of  anti-enzymes 
in  plants  has  yet  been  observed. 

The  most  remarkable  property  of  the  anti-enzymes,  in  the 
narrow  meaning  of  the  word,  is  the  rigid  specificity  of  their 
action;  this  property  they  possess  in  common  with  the  anti- 
toxines.  Whether  this  is  a  peculiarity  of  the  anti-bodies  formed 
as  protective  agents  in  the  organism,  or  whether  closer  investiga- 
tion will  show  that  the  anti-enzymes  act  specifically  only  in  the 
same  sense  as  do  the  enzymes,  cannot  at  present  be  decided. 

It  can,  however,  be  asserted  from  the  experimental  material 
at  present  available,  that  the  specificity  of  the  enzymes  does  not 
differ  fundamentally  from  that  of  other  catalysts. 


CHAPTER    VIII 
SPECIFICITY    OF   ENZYME   ACTION 

THE  question  of  the  acceleration  of  one  and  the  same  reaction 
by  different  enzyme-preparations  does  not  lend  itself  to  a 
critical  examination,  owing  to  the  impossibility  of  judging  the 
physiological  purity  of  the  preparations.  If  therefore  it  is  stated, 
for  example,  that  emulsin  hydrolyses  fats,  this  can  scarcely  have 
any  other  meaning  than  that  the  preparations  considered  con- 
tain lipases,  unless  indeed  it  is  shown  that  the  actions  on  glucosides 
and  fats  always  exhibit  parallel  courses.  Meanwhile,  the  decid- 
ing factors  in  such  cases  are  physiological  in  nature,  whilst 
the  chemist  is  concerned  more  nearly  with  the  other  problem, 
namely,  that  of  determining  what  different  reactions  are  always 
initiated  by  one  and  the  same  enzyme-preparation  and  enzyme.1 

The  investigations  of  recent  years  have  rendered  it  probable 
that  in  many  enzyme-preparations  in  which  formerly  the  presence 
of  only  one  enzyme  was  assumed,  a  number  of  different  enzymes 
exist  with  more  restricted  spheres  of  action.  It  has,  indeed, 
been  mentioned  that  emulsin  contains  at  least  five  enzymes, 
and  that  diastase  is  presumably  composed  of  a  number  of  enzymes 
which  effect  the  degradation  of  starch  in  stages. 

The  term  specificity  is  applied  to  cases  where  the  action  of 
an  enzyme  is  exerted  only  on  separate  representatives  of  a  larger 
class  of  bodies. 

Some  of  these  cases  are  understood  in  so  far  as  the  course 
of  the  chemical  change  can  be  followed.  Thus,  the  well-known 
fact  that  only  sugars  with  six  or  nine  carbon  atoms,  and  not, 
for  instance,  the  pentoses,  are  fermentable,  is  much  less  remark- 


(Zeitschr.  f.  physikal.  Chem.,  1903,  45,  513)  has  studied  the 
interesting  case  in  which  different,  inorganic  catalysts  (stannic  chloride, 
iodine  chloride,  etc.)  cause  or  accelerate  catalytically  different  reactions  of 
the  substrate  (chlorine  and  benzene). 

274 


SPECIFICITY  OF  ENZYME  ACTION  275 

able  now  that  a  representation  of  the  intermediate  products  of 
the  reaction  has  been  attained. 

The  specificity  of  the  oxydases,  to  which  attention  has  been 
repeatedly  drawn,  would  most  readily,  arise  as  a  consequence 
of  purely  chemical  facts. 

In  the  cases  where  the  specific  nature  of  the  oxydases  is 
most  pronounced,  namely,  with  the  phenolases,  it  may  be  asserted 
that  the  reactivity  of  the  simple  and  substituted  mono-,  di- 
and  tri-phenols  is  dependent  on  the  constitution  in  the  same 
way  when  "  oxydases,"  as  when  non-enzymic  manganese  com- 
pounds, form  the  oxidising  catalysts. 

To  choose  the  simplest  example:  of  hydroquinone,  pyro- 
catechol  and  resorcinol,  the  first  is  oxidised  rapidly  and  the 
second  considerably  more  slowly,  whilst  the  last  is  extremely 
resistant  to  oxidation  (cf.  Bertrand»Bull.  Soc.  Chim.,  1896, 
[iii],  15,  791). 

Another  case  is  that  of  the  Upases.  From  the  results  of 
K  a  s  1 1  e  and  Loevenhart's  measurements,  it  is  known 
that  the  ester-resolving  action  of  pancreas-extract  is  by  no  means 
exerted  on  all  esters.  Apart  from  the  fact  that  the  true  fats 
are  only  very  slightly  hydrolysed  by  this  extract,  enormous 
differences  are  observed  between  the  velocities  of  hydrolysis  of 
such  closely-allied  chemical  individuals  as  ethyl  acetate  and 
ethyl  butyrate.  A  further  list  of  similar  differences  has  been 
given  by  H.  E.  Armstrong  and  O  r  m  e  r  o  d  .  But 
with  catalytic  decompositions,  completely  analogous  behaviour  is 
shown.  For  example,  according  to  R.  Lowenherz,  the 
constants  of  hydrolysis  (with  hydrochloric  acid  as  catalyst) 
of  ethyl  formate  and  methyl  benzoate  are  in  the  ratio  of  1-1  : 
0-0003,  and  still  larger  differences  can  easily  be  found.  In  this 
connection,  it  is  to  be  noted  that  in  enzyme  reactions  very  small 
velocities  do  not  show,  since  the  extended  duration  of  the  action 
results  in  the  enzyme  becoming  inactive. 

Also  the  results  ofH.  BierryandGiaja's  experiments 
(C.  R.,  1908,  147,  268)  on  the  action  of  maltases  and  lactases  of 
various  origins  appear  to  depend  on  differences  of  degree,  and 
not  of  kind,  in  the  activities;  the  resolution  of  lactose,  lactobionic 
acid  and  lactosazone  is  effected  by  an  active  preparation,  whilst 
another  preparation  which  resolves  only  lactose  must  be  generally 
weaker. 


276  GENERAL  CHEMISTRY   OF  THE  ENZYMES 

Fischer  andAbderhalden  (H.,  1905,  46,  52;  1907, 
50,  264)  have  collected  a  large  mass  of  data  concerning  the 
power  possessed  by  pancreatic  juice  of  decomposing  polypeptides- 
The  behaviour  of  these  substances  towards  P  a  w  1  o  w  '  s  pan- 
creatic juice  is  shown  in  the  following  table: 

Hydroly  sable.  Non-hydrolysable. 

*Alanylglycine 1  Glycylalanine 

*Alanylalanine  Glycylglycine 

*Alanylleucine  A  Alanylleucine  B 

*Leucylisoserine  Leucylalanine 

Glycyl-Z-tyrosine  Leucylglycine 

*Alanylglycylglycine  Aminobutyrylglycine 

*Leucylglycylglycine  Aminobutyrylaminobutyric  acid  A 

*  Glycylleucylalanine  Aminobutyrylaminobutyric  acid  B 

*  Alanylleucylglycine  Aminoisovalerylglycine 
Dialanylcystine  Glycylphenylalanine 
Dileucylcystine  Leucylproline 
Tetraglycylglycine  Diglycylglycine 
Triglycylglycine  ester  (Cur-   Triglycylglycine 

t  i  u  s  '  s  biuret  base)  Dileucylglycylglycine 

The  hydrolysis  of  these  substances  by  acids  would,  no  doubt, 
likewise  reveal  considerable  differences  between  the  velocities. 
But,  as  K  a  s  1 1  e  and  Loevenhart's  experiments  with 
esters  show,  the  order  is  not  always  the  same  for  hydrolysability 
by  enzymes  and  by  other  catalysts.  The  lack  of  parallelism 
between  the  two  cases  may  be  due  to  enzymes  and  acids  being 
combined  to  different  extents  by  different  esters. 

The  enzymes  exhibit  the  strictest  specificity  towards  optical 
antipodes. 

After  E  .  Fischer  had  shown  how  new  optically  active 
products  are  obtained  by  purely  chemical  syntheses  (Chem. 
Ber.,  1894,  27,  3230),  the  fundamental  difference  assumed  by 
Pasteur  between  natural  and  artificial  syntheses  fell  to  the 
ground.  Four  years  later  Fischer  arrived  at  the  conclusion 
that  the  specificity  of  enzymes  towards  optical  antipodes  is  con- 
ditioned by  the  stereochemical  structure  of  the  enzymes  (H., 

*  1898,26,60). 

1  Peptides  marked  *  are  the  racemic  compounds. 


SPECIFICITY  OF  ENZYME  ACTION 


277 


He  himself,  partly  in  conjunction  with  his  collaborators, 
obtained  in  the  hydrolysis  of  the  methylglucosides  the  most 
striking  examples  of  the  influence  of  configuration  on  the  attack- 
ability  of  a  substrate. 

Both  a-  and  @-methyl-d-glucosides  are  acted  on  by  enzymes, 
but  a-  and  $-methyl-/-glucosides  remain  unchanged.  While, 
however,  a-methyl-d-glucoside  is  hydrolysed  only  by  yeast- 
enzymes,  emulsin  attacks  (3-methyl-d-glucoside  alone. 

In  general,  it  appears  that  a-glucosides  are  decomposed  by 
maltase  and  ^-glucosides  by  almond-emulsin. 


OCHs 


CH30 


HO-C-H 

r. 

H-C 
K-C  OH 


CH2OH 

a-methylglucoside. 


H-C 

H-C-OH 
I 
CH2OH 

/3-methylglucoside. 


Further  cases  in  which  enzymes  hydrolyse  stereoisomeric 
compounds  with  very  unequal  velocities  are  given  by  the  inves- 
tigations of  Fischer  and  of  Abderhalden  on  poly- 
peptides.  Some  of  their  results  are  as  follows: 


Hydrolysed. 
d-Alanyl-d-alanine 
d-Alanyl-Z-leucine 
Z-Leucyl-Meucine 
/-Leucyl-c?-glutamic  acid 


Not  hydrolysed. 
d-Alanyl-Z-alanine 
Z-Alanyl-rf-alanine 
Z-Leucylglycine 
Z-Leucyl-d-leucine 
d-Leucyl-Weucine 


Since  the  view  was  advanced  that  enzymes  act  as  optically 
active  catalysts,  numerous  cases  of  enzymic,  asymmetric  syntheses 
and  decompositions  have  been  observed. 

Asymmetric  Syntheses.  If  a  symmetrical  mole- 
cule gives  rise  to  an  asymmetric  one,  the  dextro-  and  laevo- 


278  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

modifications  are  formed  in  equal  quantities,  so  that  an  inactive, 
racemic  preparation  is  obtained.  It  is,  however,  otherwise  if  a 
molecule  which  is  already  asymmetric  is  employed  for  further 
syntheses.  If  in  one  of  the  two  optical  antipodes,  I  and  II, 
say,  in  I,  one  of  the  substituents  6  is  replaced  by  another 


radicle  r,  two  new  forms,  «i  and  @i,  may  arise.  Since  in  these 
two  forms  the  new  group  r  is  at  different  distances  from  the 
remaining  constituents  of  the  molecule,  it  is  evident  that  the 
molecules  «i  and  gi  will  be  formed  with  unequal  velocities. 

Just  as  two  diastereomeric,1  asymmetric  products  «i  and  @i 
are  formed  from  configuration  I,  so  also  II,  which  is  the  mirror- 
image  of  I,  yields  two  corresponding  diastereomerides,  a2  and  @2, 
these  being  mirror-images  to  ai  and  gi.  The  forms  I  and  II 
thus  give  the  two  pairs  of  optical  isomerides,  ai+a2  and  Pi  +  fe, 
in  different  amounts. 

As  an  example  of  an  asymmetric  synthesis  analogous  to 
those  occurring  in  the  living  organism,  the  following  case,  which 
was  investigated  by  Marckwald,  may  be  taken : 

Methylethylmalonic  acid  was  converted  into  an  acid  salt: 

C02H  C02M  CO2M 

CH3-C-C2H5  a  CH3-C-C2H5  &  C2H5-C-CH3 

C02H  C02H  C02H 

The  two  forms  of  the  acid  salt,  a  and  g, .  as  optical  antipodes' 
possess  similar  properties,  except  as  regards  the  sense  of  their 
rotation.  This  is  the  case,  however,  only  if  M  itself  is  optically 
inactive.  But  if  M  itself  be  an  optically  active  radicle,  as,  for 
instance,  if  the  acid  brucine  salt  of  the  acid  were  formed,  the 

1  Stereoisomeric  compounds  related  as  an  object  to  its  image  in  a  mirror, 
are  termed  optical  or  enantiomorphous  isomerides. 
On  the  other  hand,  stereoisomeric  compounds  which  are  not  mirror  imageSj 
one  of  the  other,  are  named  diastereoisomerides  or  dias- 
tereomerides. 


SPECIFICITY  OF  ENZYME  ACTION  279 

two  forms  a  and  @  will  no  longer  be  enantiomorphs  but  diastereo- 
merides  and  hence  will  exhibit  different  physico-chemical  be- 
haviour. If  the  mixture  of  a-  and  $-forms  is  heated  so  as  to 
remove  the  free  carboxyl  groups,  the  dextro-  and  laevo-salts 
must  be  formed  in  unequal  amounts.  The  free  valeric  acids 
obtained  by  removal  of  the  brucine  residue,  form  an  optically 
active  mixture.  According  to  Fischer's  expression,  from 
one  active  molecule  (brucine),  "  another  is  born." 

A  series  of  interesting  syntheses  was  also  carried  out  by 
McKenzie  (Journ.  Chem.  Soc.,  1904,  85,  1249)  who,  by 
reduction  of  Z-menthyl  benzoylformate  with  aluminium-amalgam, 
obtained  a  mixture  of  Z-menthyl  d-mandelate  with  a  slight  excess 
of  Z-menthyl  Z-mandelate. 

Shortly  afterwards,  by  the  reduction  of  Z-menthyl  pyruvate, 
he  succeeded  in  preparing  laevo-lactic  acid  (Journ.  Chem.  Soc., 
1905,  87,  1373).  With  the  help  of  Grignard's  reaction, 
other  asymmetric  syntheses,  such  as  that  of  laevo-atrolactinic 
acid  from  menthyl  benzoylformate  and  magnesium  menthyl 
iodide,  were  effected  (Journ.  Chem.  Soc.,  1906,  89,  365).  Mc- 
Kenzie and  Wren  prepared  the  optically  active  tartaric 
acids  by  oxidation  of  d-  and  /-bornyl  and  menthyl  fumarates 
(Journ.  Chem.  Soc.,  1907,  91,  1215). 

Of  the  investigations  in  this  direction  those  of  D  a  k  i  n 
deserve  special  mention.  After  W.  Marckwald  and 
A.  McKenzie  had  succeeded  in  showing  that  the  velocities 
of  esterification  of  two  opposed  optically  active  acids  by  one 
and  the  same  optically  active  alcohol  were  not  equal  (Chem. 
Ber.,  1899,  32,  2130;  1901,  34,  469),  Da  kin  found  (Journ.  of 
Physiol.,  1903,  30,  253)  that,  when  partially  hydrolysed  by 
lipase,  inactive  menthyl  mandelate  yields  a  strongly  dextro- 
rotatory mandelic  acid,  while  the  remaining  ester  is  correspond- 
ingly laevo-rotatory;  the  dextro-component  of  the  ester  is  hence 
hydrolysed  more  rapidly  than  the  laevo-component.1  Further 
experiments  by  this  investigator  have  led  to  a  number  of  inter- 
esting conclusions  (Journ.  of  Physiol.,  1905,  32,  199).  It  must 
be  remembered  that  two  optical  antipodes,  in  combining  with 
one  and  the  same  asymmetric  substance,  do  so  with  unequal 
velocities  and  that,  on  the  other  hand,  the  products  of  such 

1  The  hydrolysis  of  racemic  esters  has  found  practical  application  also 
in  the  preparation  of  optically  active  amino-acids  (Warburg). 


280  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

reactions  decompose  at  different  rates.  This  was  the  case  with 
D  a  k  i  n  '  s  asymmetric  ester-hydrolysis  by  lipase;  the  latter 
must  therefore  be  an  optically  active  substance  which  enters 
into  combination  with  the  ester  it  hydrolyses.  Experiment 
showed  further  that,  in  the  fractional  hydrolysis  of  a  series  of 
structurally  allied  racemic  esters,  the  components  which  are  the 
more  rapidly  attacked  always  possess  similar  configurations  but 
not  necessarily  rotations  of  the  same  sign. 

Asymmetric  hydrolysis  by  means  of  lipase  is  also  effected  if 
an  asymmetric  carbon  atom  is  present,  not  in  the  acid-  but  in 
the  alcohol-residue  of  the  ester.  D  a  k  i  n  therefore  drew  the 
conclusion  that  combination  between  enzyme  and  ester  takes 
place,  not  exclusively  at  the  acid-group  but  probably  with  the 
molecule  of  the  ester  as  a  whole. 

Finally,  a  number  of  earlier  (S  c  h  u  1  z  e  and  Bosshard, 
H.,  1886,  10,  134)  or  isolated  observations,  and  also  the  more 
recent  ones  of  A.  McKenzie  and  A  .  Harden  (Journ. 
Chem.  Soc.,  1903,  83,  424)  show  that  the  specificity  of  the  action 
of  micro-organisms  on  optical  antipodes  is  not  complete.  The 
enantiomorph  less  preferred  as  nutriment  is  also  consumed  by 
micro-organisms,  although  considerably  more  slowly  and  imper- 
fectly, and  even  in  cell-free  (active)  enzyme  solutions,  in  certain 
cases  at  least,  neither  of  the  two  forms  seems  to  remain  unat- 
tacked.  Here  also,  quantitative  measurements  of  the  relative 
attackability  of  the  antipodes  promise  valuable  results. 

While  D  a  k  i  n  '  s  experiments  dealt  entirely  with  asymmetric 
hydrolyses,  Rosenthaler  has  recently  described  (Biochem. 
Z.,  1908,  14,  238)  a  true  asymmetric  synthesis,  namely,  the  forma- 
tion of  d-benzaldehydecyanohydrin  from  benzaldehyde  and 
hydrocyanic  acid  under  the  influence  of  emulsin.  Of  his  exper- 
iments the  following  may  be  described : 

To  5  grms.  of  emulsin,  macerated  with  20  c.c.  of  water,  was  added 
0-675  grm.  of  hydrocyanic  acid;  after  an  hour,  20  grms.  of  benzaldehyde 
were  slowly  added,  the  liquid  being  kept  thoroughly  shaken  meanwhile. 
The  liquid  was  then  agitated  in  a  shaking  machine  for  an  hour,  after 
which  the  nitrile  was  isolated  and  hydrolysed,  and  the  mandelic  acid 
extracted  from  the  aqueous  solution  by  means  of  ether.  The  residue 
from  the  ether,  after  crystallisation  from  benzene,  showed  a  specific 
rotation  of  [«]/>=  -153-78°,  which  is  in  good  agreement  with  the  value 


SPECIFICITY   OF  EJNZYME   ACTIOJN 

for  mandelic  acid.     The  cyanohydrin  formed  was  free  from  the  laevo- 
form,  as  was  shown  by  hydrolysis  to  mandelic  acid. 

As  Rosenthaler  found,  in  the  emulsin  there  is  a  sub- 
stance which  conditions  the  asymmetry  of  the  synthesis  and 
another  constituent  which  accelerates  the  addition  of  hydro- 
cyanic acid  to  aldehyde  or  ketone.  The  latter  of  these  substances 
proves  to  be  a  compound  of  magnesium,  calcium  or  potassium. 
The  explanation  of  this  phenomenon  is  probably  to  be  sought 
in  Franzen's  recent  investigation  (Chem.  Ber.,  1909,  42, 
3293),  which  showed  that  aldehydes  and  ketones,  with  calcium, 
barium,  strontium  or  magnesium  cyanide,  lead  to  the  formation 
of  the  salt  of  the  corresponding  nitrile.  This  reaction  proceeds 
as  follows: 


/0-Ca-Ov 

2C6H5  •  C<f    +Ca(CN)2  =  C6H5  •  Ctt(  >CH  -  C6H5. 

XH  \CN  CW 


Of  great  interest  is  the  fact  that  Rosenthaler  (Biochem. 
Z.,  1910,  26,  1  and  28,  408)  has  succeeded  in  preparing  from 
emulsin,  besides  the  nitrile-synthesising  enzyme  (a-emulsin) 
which  we  shall  term  n  i  t  r  i  1  e  s  e  ,  an  enzyme  which  exerts 
solely  a  hydrolytic  action  and  was  named  by  him  B-emulsin. 

By  protracted  heating  at  40-45°  the  B-emulsin  is  inactivated 
completely  and  the  hydrolytic  enzyme  partially,  whilst  the 
nitrilese  remains  active. 

Suitable  treatment  with  acid  and  subsequent  neutralisation 
with  alkali  also  destroys  the  amygdalin-resolving  action  of  emulsin, 
while  the  synthetic  action  (of  the  nitrilese)  is  to  some  extent 
retained. 

The  nitrates  obtained  after  precipitating  with  copper  sul- 
phate, saturating  with  magnesium  sulphate  or  half  saturating 
with  ammonium  sulphate,  contain  no  nitrilese  but  still  hydrolyse 
amygdalin. 

In  the  decomposition  of  amygdalin,  three  enzymes  must 
hence  take  part  (cf.  p.  23):  an  amygdalase,  a  g-glucosidase 
and  a  n  i  t  r  i  1  a  s  e  which  resolves  the  mandelonitrile;  the  two 
first  are  hydrolysing  enzymes,  whilst  the  last  has  a  purely  decom- 
posing action. 

Rosenthaler  has  attempted  to  separate  his  §-emulsin 
further  into  these  three  constituents  in  the  following  manner: 


282  GENERAL  CHEMISTEY  OF  THE  ENZYMES 

1.  The  filtrates  obtained  after  precipitation  with  copper  sul- 
phate and  half  saturating  with  ammonium  sulphate  contained 
all  the  three  enzymes. 

2.  The   filtrate   obtained   after   saturating   with   magnesium 
sulphate  contained  hydrolysing  enzyme,  but  no  nitrilase. 

No  other  means  could  be  discovered  of  separating  the  hydror 
lysing  enzyme  from  that  which  decomposes  the  nitrile. 

No  satisfactory  theoretical  treatment  of  the  co-existence 
and  co-operation  of  a  synthetic  and  a  decomposing  enzyme  has, 
in  the  author's  opinion,  yet  been  advanced.  Mention  must, 
however,  not  be  omitted  of  the  theory  developed  by  F  a  j  a  n  s 
(Dissertation,  Heidelberg,  1910;  Zeitschr.  f.  physikal.  Chem., 
1910,  73,  25). 

An  interesting  case,  closely  related  to  the  above,  has  been 
described  by  B  r  e  d  i  g  and  F  a  j  a  n  s  (Chem.  Ber.,  1908,  41, 
752).  The  two  optically  active  camphocarboxylic  acids,  which 
readily  decompose  into  camphor  and  carbon  dioxide  when  heated : 

CioHi50  •  CO2H  •=  CioHiG0+C02, 

do  so  with  different  velocities  when  they  are  dissolved  either  in 
pure  nicotine  or  in  a  solvent  containing  nicotine.  The  follow- 
ing results  were  obtained: 

Velocity  of  liberation  of  carbon  dioxide  in  nicotine  at  70°. 


Per  1  grm.  dextro-acid. 


fed 


Dissolved  in   3  c.c.  nicotine  .  0-00493 
"  5  0-00493 

"          10          "  .  0-00479 


Mean..  .  0-00488 


Per  1  grm.  laevo-acid. 

Dissolved  in   5  c.c.  nicotine  .  0-00436 

"  5          "  .  0-00444 

10          "  .  0-00421 


Mean..  .   0-00434 


Hence  in  nicotine  as  solvent,  the  d-acid  decomposes  about 
13%  more  rapidly  than  the  Z-acid.  Salt-formation  evidently 
takes  place  between  the  active  acid  and  the  active  base,  the 
diastereomeric  bodies  thus  formed  differing  in  their  chemical 
behaviour. 

In  the  case  studied  by  B  r  e  d  i  g  ,  the  carbon  dioxide  liberated 
is  evolved,  so  that  the  nicotine  previously  combined  with  the 
camphocarboxylic  acids  becomes  free  after  the  decomposition 
and  can  form  salt  with  fresh  quantities  of  acid. 


SPECIFICITY  OF  ENZYME  ACTION  283 

The  difference  between  the  experiments  of  Marckwald 
and  those  of  B  r  e  d  i  g  consists  in  the  employment  by  the  latter 
of  a  weaker  base.  The  reaction  studied  by  B  r  e  d  i  g  hence 
assumes  the  character  of  a  catalytic  process. 

Nevertheless,  both  the  results  obtained  by  Marckwald 
and  those  of  B  r  e  d  i  g  support  the  view,  first  expressed  by 
Fischer,  that  the  enzymes  are  optically  active  catalysts. 
Their  mode  of  action  may  apparently  be  expressed  as  follows: 

By  combination  with  the  racemic  substrate,  the  optically 
active  enzymes  give  rise  to  diastereomeric  substances,  which 
decompose  with  different  velocities  and  hence  result  in  the  forma- 
tion of  optically  active  material. 

E  .  Fischer  made  an  interesting  experiment  to  ascertain 
if  two  oppositely  active  acids,  d-  and  Z-camphoric  acids,  hydro- 
lyse  cane-sugar  with  different  velocities,  but  the  result  was  neg- 
ative. The  probability  of  the  assumption  that  catalysing  acids, 
like  enzymes,  combine  with  the  substrate,  suggests  the  extension 
of  this  experiment  and  the  making  of  others  in  which  a  substrate 
consisting  of  two  enantiomorphs  shall  be  decomposed  by  an 
optically  active  catalyst.  The  author  has  been  occupied  with 
such  experiments  for  several  years.  It  is  evident  that  all  facts 
are  of  value  which  furnish  further  knoweldge  of  the  union  between 
enzyme  and  substrate. 

By  the  representation  of  the  key  fitting  the  lock,  that  is,  by 
the  hypothesis  that  the  enzymes  are  optically  active  catalysts, 
the  enzymes  are  brought  into  close  relation  with  other  catalysts. 
The  development  of  this  hypothesis  is  undoubtedly  one  of  the 
most  important  aims  of  the  chemistry  of  the  enzymes. 

CONCLUSION 

WHAT  then  can  be  given  as  the  results  of  the  investigation 
of  the  enzymes? 

As  regards  the  chemical  nature  of  the  enzymes,  the  result 
of  our  survey  is  but  negative,  inasmuch  as  the  analyses  and  chem- 
ical reactions  of  various  enzyme  preparations  furnish  no  evidence 
in  support  of  the  statement — often  found  in  the  literature  1— 
that  enzymes  are  protein  substances;  further  there  is  nothing 
to  indicate  that  all  enzymes  belong  to  a  single  class  of  substances. 

1  See,  for  instance,   V  e  r  n  o  n  ,  Ergeb.  der  Physiol.,  1910,  9,  227. 


284  GENERAL   CHEMISTRY  OF  THE  ENZYMES 

On  the  other  hand,  no  fact  is  known  which  definitely  disproves 
the  protein  character  of  any  of  the  hydrolytic  enzymes,  since  the 
results  indicating  the  failure,  of  enzyme  solutions  to  give  protein 
reactions  do  not  give  the  concentrations  of  the  solutions  and  have 
not  been  sufficiently  controlled  by  means  of  similarly  dilute  solu- 
tions of  undoubted  proteins. 

All  that  has  yet  been  stated  concerning  the  chemical  con- 
stitution is  mere  supposition.  Better  than  from  the  purely 
chemical  investigations,  we  could,  from  the  physico-chemical 
measurements  of  thermo-sensibility,  i.e.,  from  the  inactivation 
constants,  attempt  to  derive  certain  relations  with  the  proteins, 
the  denaturation  of  these  by  heat  bearing  a  close  resemblance 
to  that  of  the  enzymes.  But  perhaps  the  saponins  exhibit  still 
more  marked  analogies  to  those  remarkable  colloidal  poisons, 
the  physico-chemical  behaviour  of  which  is  still  insufficiently 
investigated. 

The  view  that,  for  the  development  of  their  activity  towards 
the  substrate,  certain  enzymes  require  the  presence  of  other 
substances — co-enzyme,  acid,  etc. — which  are  classed  together  as 
activators,  has  resulted  in  a  thorough  qualitative  investigation 
of  the  chemistry  of  enzyme-action,  and  the  consideration  of 
these  activators  is  of  the  utmost  importance  to  the  chemico- 
dynamic  study  of  the  enzymes. 

The  many  deviations  of  the  best-known  enzymes,  e.g., 
invertase,  from  the  simple  relations  required  by  the  law  of  mass 
action,  had  led  to  a  formal  treatment  of  enzymic  reactions,  but 
the  results  of  this  correspond,  by  no  means,  with  the  amount 
of  labour  expended.  Only  in  the  most  recent  times  has  the  nec- 
essary revision  of  the  earlier  experiments  been  commenced;  never- 
theless, it  cannot  be  regarded  as  premature  to  assert  that  the 
reactions  induced  by  enzymes — the  enzymic  hydrolyses  being 
here  especially  referred  to — follow  the  laws  which  hold  generally 
for  catalytic  reactions  in  solution  and  are  deducible  theoretically 
from  the  law  of  mass-action.  Correspondence  of  the  time-law 
with  that  for  unimolecular  reactions,  and  proportionality  between 
concentration  of  the  enzyme  and  velocity  of  reaction  were  fre- 
quently observed.  Where  these  relations  are  not  obeyed,  the 
disturbing  influence  exerted  by  the  products  of  the  reaction 
either  is  known  with  certainty  or  may  be  assumed  with  a  high 
degree  of  probability.  In  individual  cases,  the  ultimate  cause 


SPECIFICITY  OF  ENZYME  ACTION  285 

of  this  disturbance  is  still  uncertain;  sometimes  it  must  be  the 
enzymes  themselves,  but  in  many  instances  the  activators, 
which  are  combined,  the  latter  case  appearing  to  be  the  more 
common.  In  any  case,  we  have  seen  that  a  simple  explanation 
is  forthcoming  for  S  c  h  u  t  z  '  s  rule;  relations  similar  to  that  of 
S  c  h  ii  t  z  can  also  occur  with  inorganic  catalysts,  one  of  the 
best-known,  apparent  peculiarities  of  enzymic  reactions  thus 
falling  to  the  ground.  For,  that  the  numerous  "  laws  "  such  as 

7      1          a+x 


etc.,  possess  neither  real  significance  nor  validity  may  be  regarded 
as  an  established  and  pleasing  fact. 

The  enzymes  are,  therefore,  catalysts.  Do  they,  like  the 
inorganic  catalysts  of  the  best-known  reactions,  for  instance, 
hydrolysis  of  esters,  leave  the  equilibrium  of  the  reaction 
unchanged?  Perhaps,  under  some  circumstances  and  more  fre- 
quently than  now  appears  to  be  the  case,  they  do  so,  but  it  can 
be  stated  with  certainty  that  this  does  not  always  happen,  and 
the  conception  of  a  catalyst  must  hence  be  made  more  compre- 
hensive than  experience  of  non-enzymic  reactions  requiies.1 
But  this  leads  to  no  disagreement  with  the  principles  of  chemical 
dynamics  or  with  the  fundamental  laws  of  thermodynamics,  it 
being  only  necessary  to  assume  that  a  considerable  proportion 
of  the  enzyme  can  combine  with  the  substrate  or  with  the  pro- 
ducts of  the  reaction.  The  equilibrium  must  evidently  depend  on 
the  concentration  of  these  new  molecules,  enzyme-substrate  or 
enzyme-reaction  product,  and  any  circumstance  altering  this 
concentration  alters  also  the  equilibrium  or  the  stationary  con- 
dition of  the  reaction. 

The  configuration  of  the  substrate,  the  spatial  arrangement 
of  its  atoms,  is,  as  was  seen  in  the  preceding  chapter,  of  deter- 
mining importance  for  the  occurrence  of  an  enzymic  reaction. 
Emil  Fischer  has  given  us  the  theory  for  these  facts,  the 
underlying  assumption  being  that  an  enzyme  is  an  optically 
active  catalyst.  This  forms,  with  the  two  components  of  a  racemic 
mixture,  "  active  molecules  "  which  are  not  enantiomorphous 
but  diastereomeric  products,  differing  in  their  chemical  properties 

1  Cf.  Taylor,  Journ.  of  Biol.  Chem.,  1910,  8,  503. 


286  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

and  hence  leading  to  the  decomposition  of  the  components  with 
unequal  velocities.  Thus,  what  formerly  appeared  to  char- 
acterise the  catalysts  of  living  matter  presents  itself,  in  the  light 
of  this  theory,  as  a  consequence  of  our  views  of  the  configuration 
of  molecules. 

But  what  is  the  nature  of  the  combination  between  the 
enzyme  and  the  substance  succumbing  to  chemical  attack, 
and  how  does  the  living  organism  maintain  the  equilibrium 
between  enzyme  and  substrate  so  important  to  its  existence? 
Here  we  meet  the  great  riddle  of  the  formation  of  enzymes 
and  anti-enzymes  in  the  organism  which  opens  out  a  region  of 
investigation  of  immeasurable  breadth. 


APPENDIX 


PRACTICAL    METHODS 

IN  what  follows,  a  short  description  is  given  of  those  methods 
of  investigating  enzyme-preparations  and  of  following  enzymic 
decompositions  which  have  been  or  might  be  generally  applied 
either  in  medicinal  practice  or  in  industrial  work. 

Scientific  investigation  of  enzymic  reactions  has  often  been 
effected  with  the  acid  of  physico-chemical  methods,  but  a  detailed 
account  of  these  would  occupy  too  much  space  here,  so  that 
reference  must  be  made  to  the  special  literature  of  the  subject. 
Particular  mention  may  be  made  of : 

O  s  t  w  a  1  d  and  Luther  :  Manual  of  Physico-chemical 
Measurements. 

W.  A  .  R  o  t  h  :  Exercises  in  Physical  Chemistry,  London, 
1909. 

Hamburger  :  Osmotischer  Druck  und  lonenlehre  in  den 
medizinischen  Wissenschaften,  Wiesbaden,  1902-1904. 

Also  shorter  references  by: 

H.  Friedenthal,  L.  Michaelis,  etc.,  in  Abder- 
halden's  Handbuch  der  biochemischen  Arbeitsmethoden,  Berlin, 
1910-1912. 

A  .  K  a  n  i  t  z  ,  in  Oppenheimer's  Handbuch  der  Biochemie 
des  Menschen  und  der  Tiere,  Band  I,  1908. 

As  regards  the  methods  of  obtaining  enzymes,  these  have 
been  given  in  the  first  chapter  of  this  book,  where  also  the 
preparation  of  the  separate  enzymes  in  a  pure  state  has  been 
described. 

It  is  only  necessary  here  to  call  attention  to  the  fact  that 
enzymes  which  exert  their  actions  within  the  walls  of  a  cell  are 
often  either  not  at  all  or  only  very  incompletely  extractable,  so 

287 


288  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

long  as  the  cell- wall  is  living  and  uninjured;  the  dead  cell-wall 
allows  of  much  readier  passage  to  the  enzyme,  which  can  thus 
be  obtained  in  several  ways. 

1.  The  enzymic  material  is  dried  as  rapidly,  and  at  as  low  a 
temperature,  as  possible.     By  this  treatment,  the  cell-walls  are 
rendered,  in  st)me  cases,  more  permeable  and  in  others,  more 
easily  ruptured;    these  effects  are  enhanced  if  the  dehydrated 
cells  are    heated     at     about     50-70°     [cf.     E.     Fischer's 
method  of  preparing  invertase   (Chem.  Ber.,   1894,   27,   2985); 
and  Wiechowski   and  Wiener's  method  (Hofm.  Beitr., 
1907,  9,  232)  for  preparing  from  the  kidneys  the  enzyme  which 
oxidises  uric  acid]. 

2.  The  finely-divided  material,  e.g.,  yeast,  after  being  freed 
mechanically  from  water,   is  introduced  into  absolute   alcohol 
or  anhydrous  acetone.     Here  also,  the  dehydration  of  the  mate- 
rial by  the  organic  solvent  should  be  as  rapid  as  possible. 

3.  The  cell-walls  may  be  destroyed  by  autolysis  (see  O  '  Sul- 
livan   and  Tompson's    method,  p.  26). 

4.  The  cells,  while  still  living,   are  ruptured  mechanically. 
The  method  so  successfully  employed  by  Buchner    (see  p. 
56)    is   well   known.     Rowland     (Journ.    of   PhysioL,    1901, 
27,  53)  gave  a  somewhat  different  method,  in  which  a  mixture 
of  the  cells  with  sand  is  made  to  assume  a  vigorous  rotatory 
motion;   the  action  resembles  that  of  a  sand-blast. 

Bacteria,  soft  organs,  etc.,  can  be  hardened  by  cooling  in 
liquid  air  and  are  then  readily  broken  up. 

The  analytical  methods  employed  may  now  be 
mentioned. 

L  i  p  a  s  e  s  .  Pancreas-lipase  is  very  suitably  tested  by 
means  of  an  aqueous  emulsion  of  egg-yolk,  as  in  the  work  of 
V  o  1  h  a  r  d  and  others.  The  quantity  of  fat  hydrolysed  in  time 
t  is  measured  and  the  total  amount  hydrolysable  then  cal- 
culated. 

To  this  end,  the  egg-yolk  emulsion  containing  the  lipase  is 
extracted  with  ether:  (I)  An  aliquot  part  (50  c.c.)  of  the  ethereal 
extract  is  titrated  after  addition  of  50  c.c.  of  alcohol  and  then 
hydrolysed  with  10  c.c.  of  normal  sodium  hydroxide  solution, 
the  salts  of  the  fatty  acids  being  decomposed  after  24  hours  by 
means  of  10  c.c.  of  normal  sulphuric  acid.  (II)  The  fatty  acids 


APPENDIX  289 

obtained  by  hydrolysis  are  estimated  by  titration  and  the  per- 
centage x  of  fatty  acid  split  off  by  the  enzyme  calculated  by  the 
formula,  I  :  I+II  =  z  :  100. 

In  shaking  the  fat-emulsion  with  ether,  more  rapid  separation 
is  effected  if  2-10  c.c.  of  alcohol  are  added  to  the  ether. 

In  this  connection  see  Stade,  Hofm.  Beitr.,  1902,  3,  291, 
and  E  n  g  e  1 ,  Hofm.  Beitr.,  1905,  7,  78. 

Esterases  of  lower  esters.  Ethyl  butyrate  is 
best  employed  as  substrate.  The  course  of  the  reaction  is  fol- 
lowed by  direct  titration  or  by  observation  of  the  change  of  the 
electrical  conductivity. 

Vegetable  lipases.  Ricinus  seeds  are  skinned,  freed 
from  oil  by  pressing  and  treating  the  pressed  cake  with  ether, 
and  finely  ground.  The  seed-juice  formed  is  separated  from  the 
inactive  constituents  of  the  seed  in  a  centrifuge.  This  juice  is 
allowed  to  stand  for  24  hours,  during  which  time  the  enzymic 
emulsion,  in  which  the  acid  (lactic)  necessary  for  activation  is 
formed,  collects  at  the  surface  and  can  be  removed.  One  hun- 
dred grms.  of  oil  and  0-2  grm.  of  manganous  sulphate  are  stirred 
up  with  this  emulsion  (5-10  grms.)  and  the  mixture  left.  Here 
also  the  lipolysis  can  be  followed  by  titration. 

A  m  y  1  a  s  e  s  .  For  the  estimation  of  the  diastatic  power  of 
malt  for  brewery  purposes,  L  i  n  t  n  e  r  (Zeitschr.  f.  prakt. 
Chem.,  1886,  34,  386)  gave  the  following  method,  which,  in  prac- 
tised hands,  gives  good  results. 

Separate  volumes;  of  0-1,  0-2,  0-3,  ....  1-0  c.c.  of  malt 
extract  [25  grms.  of  the  ground  malt +500  c.c.  of  water,  allowed 
to  stand  at  21°  (70°  F.)  for  3  hours  and  then  filtered  bright] 
are  added  to  a  series  of  10  test-tubes,  each  containing  10  c.c.  of 
2%  soluble  starch  solution,  the  contents  of  each  tube  being  well 
mixed.  After  exactly  1  hour's  rest  at  21°,  5  c.c.  of  Fehling's 
solution  are  mixed  with  the  liquid  in  each  tube  and  the  tubes 
then  immersed  in  a  boiling  water-bath  for  exactly  10  minutes, 
after  which  the  precipitate  is  allowed  to  settle.  If  the  Fehling's 
solution  in  the  tube  containing  0-1  c.c.  of  malt  extract  is  just 
completely  reduced,  the  diastatic  power  of  the  malt  is  taken  as 
100;  if  that  in  the  one  containing  0-2  c.c.  of  the  extract,  the 
diastatic  power  is  50,  and  so  on.  A  more  exact  result  may  be 


290  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

obtained,  if  necessary,  by  taking  0-1,  0-15,  0-2,  0-25,  etc.,  c.c. 
of  malt  extract  for  the  series  of  tubes. 

The  cold-water  malt  extract  itself  contains  a  small  amount 
of  sugars  which  reduce  Fehling's  solution;  the  extent  of  this 
reduction  may  be  determined  by  direct  experiment,  but  for  all 
ordinary  purposes  it  is  sufficiently  accurate  to  deduct  1*5  from 
the  value  obtained  for  the  diastatic  power  in  the  manner  described 
above. 

The  following  modification  of  the  above  method  has  been 
recently  devised  by  Ling  and  is  widely  used :  3  •  0  (or  1  •  0,  2  •  0, 
or  4-0,  according  to  the  expected  diastatic  power)  c.c.  of  the 
malt  extract  (prepared  as  already  described)  are  added  to  100 
c.c.  of  2%  soluble  starch  solution  in  a  200-c.c.  flask,  the  mixture 
being  kept  at  21°  (70°  F.)  for  1  hour.  At  the  end  of  this  time, 
20  c.c.  of  N/10-sodium  hydroxide  solution  are  added  and  the 
liquid  made  up  to  200  c.c.  with  water.  After  mixing,  this  solu- 
tion is  introduced  into  a  burette  and  gradually  run  into  5  c.c. 
of  Fehling's  solution  diluted  with  a  little  water  and  kept  boiling; 
this  is  continued  until  the  solution  just  loses  its  blue  colour  or 
fails  to  give  a  brown  coloration  with  a  drop  of  ferrous  thiocyanate 
solution  on  a  white  tile.  If,  say,  25  c.c.  of  the  liquid  (100  c.c. 
of  which  corresponds  with  1  grm.  of  soluble  starch  and  1-5  c.c. 
of  malt  extract)  are  required  tc  reduce  5  c.c.  of  Fehling's  solution, 
the  diastatic  power  of  the  malt  will  be 

1000    =26-7. 


25X1-5 

This  method  gives  excellent  results,   in  exact  agreement  with 
those  given  by  L  i  n  t  n  e  r  's  method. 

With  preparations  of  diastase,  L  i  n  t  n  e  r  dissolves  0  •  2-0  •  5 
grm.  (according  to  the  activity)  in  50  c.c.  of  water  and  adds  0-1, 
0-2, .  .  .1-0  c.c.  of  this  solution  to  a  series  of  10  test-tubes,  each 
charged  with  10  c.c.  of  2%  starch  solution.  The  subsequent 
procedure  is  exactly  similar  to  that  employed  in  the  case  of  malts. 

A  soluble  starch  which  can  be  readily  prepared  with  constant 
properties,  is  obtained  by  allowing  potato  starch  to  remain 
under  7  •  5%  hydrochloric  acid  solution  for  7  days  at  the  ordinary 
temperature,  then  removing  the  acid  completely  by  washing 
with  cold  water,  and  drying  the  starch  in  the  air.  This  method 


APPENDIX  291 

yields  a  product  dissolving  readily  in  hot  water  to  a  clear  solution 
(cf.  G.C.Jones,  Journ.  Inst.  of  Brewing,  1908,  14,  13). 

The  determination  of  the  velocity  of  hydrolysis  by  oxidation 
of  the  liquid  with  F  e  h  1  i  n  g  '  s  solution  (cf .  Wroblewski, 
H.,  1898,  24,  173)  is,  indeed,  the  most  reliable  of  the  methods 
yet  developed,  although  it  is  still  capable  of  improvement. 

A  gravimetric  method,  which  seems  to  give  good  results, 
has  recently  been  proposed  by  Sherman,  Kendall  and 
Clark  (Journ.  Amer.  Chem.  Soc.,  1910,  32,  1073),  who  have 
also  compared  the  older  methods. 

A  number  of  other  methods  are  based  on  the  colorations 
produced  by  iodine  in  starch  and  dextrin  solutions.  Of  the 
earlier  methods,  those  of  D  e  t  m  a  r  (H.,  1882,  7,  1)  and 
Roberts  (Proc.  Roy.  Soc.,  1881,  32,  145)  may  be  referred  to, 
whilst  the  following  method,  given  by  Wohlgemuth 
(Biochem.  Z.,  1908,  9,  1),  deserves  special  mention. 

To  each  of  a  series  of  test-tubes  containing  different  quanti- 
ties of  the  enzyme  solution  to  be  tested  are  added  5  c.c.  of  1% 
starch  solution,  each  tube  being  placed  immediately  in  a  wire 
basket  standing  in  ice-water,  so  as  to  prevent  the  slightest  enzyme 
action.  When  all  the  tubes  are  ready,  the  wire  basket  contain- 
ing them  is  transferred  to  a  water-bath  at  40°;  by  this  means, 
the  enzyme  action  in  each  tube  begins  at  the  same  moment. 
After  30  or  60  minutes,  the  basket  is  placed  again  for  a  short  time 
in  ice-water,  so  that  all  the  actions  are  interrupted  at  the  same 
instant.  The  strength  of  the  enzyme  solution  is  then  deter- 
mined as  follows: 

All  the  tubes  are  filled  with  water  up  to  within  about  the 
thickness  of  the  finger  from  the  top,  and  to  each  is  added  a  drop 
of  decinormal  iodine  solution,  the  liquid  being  then  mixed. 
Different  colorations — dark  blue,  bluish-violet,  reddish-yellow 
and  yellow — are  thus  obtained.  The  tubes  showing  a  yellow  or 
reddish-yellow  colour  contain — disregarding  further  degradation 
of  the  starch  to  maltose  or  isomaltose  and  dextrose — only  achro- 
odextrin  or  erythrodextrin,  the  bluish-violet  ones  contain  a  mixture 
of  erythrodextrin  and  starch,  whilst  those  with  a  dark-blue 
colour  contain  mainly  unaltered  starch.  As  the  lower  limit  of 
the  activity  (limes)  Wohlgemuth  takes  the  first  tube  in 
which  the  blue  colour  cannot  be  detected,  i.e.,  the  one  showing 


292  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

a  violet  colour.  In  the  preceding  tube,  the  whole  of  the  starch 
is  broken  down  to  the  dextrin  stage  at  least;  from  this,  calcula- 
tion is  made  of  the  number  of  c.c.  of  1%  starch  solution  degraded 
to  dextrin  by  1-0  c.c.  of  enzyme  in  the  time  employed  for  the 
experiment. 

Suppose  that  the  tube  which  has  become  just  colourless 
contained  0*02  c.c.  of  saliva;  this  amount  was  then  able  to  trans- 
form 5  c.c.  of  1%  starch  solution  into  dextrin  in  30  minutes, 
so  that  1  c.c  of  saliva  corresponds  with  250  c.c.  of  1%  starch 
solution.  To  indicate  the  diastatic  power  of  1  c.c.  of  the  enzyme 
solution,  Wohlgemuth  contracts  the  word  diastase  to  D 
and  suggests  that  the  temperature  and  duration  be  given  for 
each  experiment.  The  result  of  the  above  determination  would 
then  be  stated  thus:  D3Q,  =  250.  To  calculate  the  diastatic 
power  from  an  experiment  in  which  tube  No.  5  was  taken  as  the 
lower  limit,  while  tube  No.  4,  containing  0-0125  c.c.  of  saliva, 
was  coloured  red  (see  the  scale  of  colour  given,  1  o  c  .  c  i  t  .)  , 
we  must  proceed  as  follows: 

In  30  minutes,  0-0125  c.c!  degrades  5  c.c.  1%  starch  solution. 
In  30  minutes,  1-0000  c.c.  degrades  400  c.c.  1%  starch  solution, 


Other  colorimetric  iodine  methods  are  given  by  J  u  n  g  k 
and  by  Johnson  (Journ.  Amer.  Chem.  Soc.,  1908,  30,  798). 
A  criticism  of  these  methods  will  be  found  in  Sherman, 
Kendall  and  C  1  a  r  k  's  paper  (1  o  c.  cit.). 

G  1  i  n  s  k  i  and  Walther  (Pawlow,  Arb.  d.  Ver- 
dauungsdrlisen,  Wiesbaden,  1898)  have  applied  M  e  1  1  '  s  method 
to  the  estimation  of  diastase.  Narrow  glass  tubes,  open  at  both 
ends,  are  filled  with  starch-paste  and  immersed  in  the  enzyme 
solution,  the  length  of  the  dissolved  cylinder  of  starch  being  meas- 
ured after  a  certain  lapse  of  time.  The  velocity  of  this  reaction 
is  evidently  influenced  considerably  by  the  diffusion  of  the  enzyme 
to  the  surface  of  the  starch  and  by  the  rate  at  which  the  hydrolytic 
products  are  removed  from  this  surface;  movement  of  the  liquid 
or  of  the  tubes  in  the  liquid  has,  therefore,  considerable  effect. 
On  the  other  hand,  this  method  of  procedure  measures  the  liquefac- 
tion of  the  starch-paste  which  is  not  a  direct  measure  of  the  sac- 
charifying action  of  the  amylase.  The  method  is  therefore 
applicable  only  in  special  cases. 


APPENDIX  293 

According  to  E  d  .  M  u  1 1  e  r  (Zentralbl.  f.  inn.  Med.,  1908), 
the  use  of  plates  of  starch-paste  presents  certain  advantages. 

Enzymes  of  the  Disaccharides  and  G 1  u- 
c  o  s  i  d  e  s  .  In  these  cases,  the  change  of  the  optical  rotation 
affords  a  simple  and  accurate  method  of  following  the  reaction. 
As  was  pointed  out  on  p.  159,  it  is  essential  to  destroy  the  muta- 
rotation  of  glucose;  this  is  best  effected  by  the  addition  of  soda 
immediately  before  reading  the  rotation. 

Another  method  consists  in  determining  the  reducing  power 
of  the  solution.  This  is  carried  out  with  Fehling's  solution  in 
one  of  a  number  of  ways,  of  which  that  of  Bertrand  is  one 
of  the  most  accurate  and  convenient. 

Bertrand  (Bull.  Soc.  Chim.,  1906,  35,  1285)  boils  the  sugar 
solution  to  be  tested  with  Fehling's  solution  of  definite  com- 
position for  three  minutes,  the  time  being  reckoned  from  the  instant 
when  the  first  bubbles  form.  The  precipitated  cuprous  oxide  is  filtered 
on  an  asbestos  filter  and  washed  with  hot  water.  The  cuprous  oxide 
remaining  in  the  E  r  1  e  n  m  e  y-e  r  boiling  flask  and  also  that  collected 
on  the  filter  are  dissolved  in  a  solution  of  ferric  sulphate  in  sulphuric 
acid,  the  following  reaction  occurring: 

Cu20  +Fe2(S04)3  +H2S04  =  2CuS04  +H20  +2FeS04. 

The  ferrous  salt  is  titrated  with  permanganate  solution,  standardised 
by  means  of  ammonium  oxalate.  Bertrand  has  prepared  tables 
for  the  most  important  reducing  sugars,  so  that  the  amount  of  sugar 
can  readily  be  obtained  from  that  of  the  cuprous  oxide  formed. 

The  solutions  employed  have  the  following  compositions: 

F~e  hling's  solution.  Iron  solution. 

Copper  sulphate 40  grms.  Ferric  sulphate 50  grms. 

Rochelle  salt 200  Sulphuric  acid 200 

Sodium  hydroxide 150     "  Water  to 1  litre 

Water  to 1  litre 

Permanganate    solution 
5  grms.  potassium  permanganate  per  litre. 

The  iron  solution  should  not  reduce  the  permanganate.  If  this 
does  occur,  the  permanganate  solution  is  gradually  added  to  the  iron 
solution  until  the  latter  assumes  a  slight  pink  colour;  it  is  then  ready 
for  use. 


294  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

When  a  sugar  solution  is  to  be  titrated,  20  c.c.  of  it  are  intro- 
duced into  an  Erlenmeyer  flask  of  125-150  c.c.  capacity.  The 
amount  of  sugar  in  this  volume  of  the  solution  is  preferably 
0-010-0-090  grm.  and  should  not  exceed  0-100  grm. 

To  the  sugar  solution  are  added  20  c.c.  of  the  copper  sulphate 
solution  and  20  c.c.  of  the  alkaline  tartrate  solution — which 
are  best  kept  separate — the  liquid  being  then  boiled  for  just  3 
minutes  after  the  first  appearance  of  bubbles. 

For  separating  the  cuprous  oxide,  use  is  made  of  a  Gooch 
crucible  packed  with  asbestos  and  fitted  as  usual  to  a  pump- 
flask.  The  precipitate  is  washed  with  hot  water  and  as  little  as 
possible  of  it  collected  on  the  filter.  When  the  washing  is  complete, 
the  main  quantity  of  cuprous  oxide  in  the  Erlenmeyer  flask  is 
dissolved  in  a  known  volume  of  the  ferric  sulphate  solution; 
the  oxide  changes  from  bright  red  to  blue-black  and  finally 
yields  a  clear,  pale-green  solution.  This  is  then  poured  through 
the  filter  to  dissolve  the  remaining  cuprous  oxide,  more  ferric 
sulphate  solution  being  added  if  necessary.  When  all  the  oxide 
is  dissolved,  the  Erlenmeyer  flask  and  the  filter  are  washed  with 
water  and  the  combined  liquids  titrated  in  the  pump-flask  with 
the  permanganate  solution.  The  change  in  colour  from  green 
to  pink  is  extremely  sharp. 

The  equation  given  above  shows  that  2  atoms  of  copper 
correspond  with  2  mols.  of  ferrous  sulphate  and  hence  with 
2  atoms  of  iron  to  be  oxidised  by  the  permanganate.  The 
iron-titre  of  the  permanganate  has  thus  only  to  be  multiplied 
by  the  ratio, 

63-6  :  55-9-1-1377, 

in  order  to  obtain  the  amount  of  copper,  the  corresponding 
quantity  of  the  sugar  being  then  given  by  the  tables. 

The  permanganate  solution  is  standardised  as  follows:  a 
weighed  quantity  of  about  0-25  grm.  of  ammonium  oxalate  is 
dissolved  in  a  beaker  in  50-100  c.c.  of  water  and  1-2  c.c.  of  pure 
sulphuric  acid.  The  liquid  is  heated  to  60-80°  and  the  perman- 
ganate solution  run  in  from  a  burette  until  a  pink  colour  is  obtained. 

One  molecule  of  ammonium  oxalate,  (NH4)  20264  -HH^O 
(mol.  wt.,  142-1)  corresponds  with  2  Fe  and  hence  with  2  Cu. 

r»O      £>  vx  O 

Multiplication  of  the  weight  of   oxalate  by  ~142-1  '  *'6''  ^ 


APPENDIX  295 

0-8951,  gives  the  quantity  of  copper  corresponding  with  the 
volume  of  permanganate  solution  required  to  produce  the  pink 
coloration.  One  litre  of  the  permanganate  solution  will  cor- 
respond with  about  10  grms.  of  copper. 

The  tables  to  be  used  with  this  method  are  given  at  the  end 
of  this  section  (pp.  306-311). 

I.  B  a  n  g  (Biochem.  Z.,  1906,  2,  271)  has  described  a  new 
method  for  the  estimation  of  reducing  sugars  which  may  be  applied 
to  the  study  of  enzymic  processes.  It  depends  on  the  fact  that, 
in  presence  of  potassium  thiocyanate,  cuprous  oxide  separates 
as  white,  insoluble  copper  thiocyanate.  The  description  of  the 
method  is  readily  accessible  and  will  not  be  given  in  detail  here. 

E  m  u  1  s  i  n  .  In  discussing  the  possible  methods  for  measur- 
ing the  decomposition  of  amygdalin,  A  u  1  d  points  out  that  the 
estimation  of  the  sugar  liberated  is  affected  by  a  number  of 
considerations.  Here  also,  the  influence  of  mutarotation  must 
be  removed.  He  employs,  therefore,  in  the  investigation  referred 
to  on  p.  173,  D  u  n  s  t  a  n  and  Henry's  titrimetric  method 
(Proc.  Roy.  Soc.,  1903,  72,  287)  of  estimating  the  free  hydro- 
cyanic acid  by  means  of  standard  iodine  solution. 

The  reaction  proceeds  according  to  the  equation: 

HCN+I2  =  CNI+HI. 

Excess  of  sodium  bicarbonate  is  employed  to  combine  with  the 
hydriodic  acid  formed. 


Proteolytic  Enzymes 

On  account  of  the  great  importance  of  measurements  of 
digestive  action  to  pure  enzymology  and  also  to  practical  medicine, 
the  number  of  communications  dealing  with  methods  employed 
in  these  measurements  is  very  large. 

1 .  An  optical  method  was  employed  byE.Schiitz 
(compare  p.  175).  He  removed  the  undigested  protein  from 
the  peptic  albumin  solutions  and  estimated  the  quantity  of 
peptone  formed  from  the  optical  rotation  of  the  residual  liquid. 

S  c  h  ii  t  z  and  H  u  p  p  e  r  t  also  used  this  polarimetric 
method  (see  p.  179)  whilst  Abderhalden  and  K  o  e  1  k  e  r 


296  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

have  recently  studied  the  action  of  tryptic  enzymes  on  optically 
active  polypeptides  by  direct  measurement  of  the  change  of 
rotation  of  the  solution. 

Ib.  Obermayer  and  Pick  (Hofm.  Beitr.,  1905,  7,  331) 
attempted  to  apply  the  alteration  of  the  refractive  index  to  the  study 
of  enzymic  reactions.  But  this  magnitude  changes  only  in  the  case 
of  tryptic  digestion. 

Griitzner  (Pfliig.  Arch.,  1874,  8,  452;  1905,  106,  463) 
has  devised  a  colorimetric  method.  Fibrin,  which  has  been 
softened  by  immersion  in  0-1%  hydrochloric  acid  containing 
carmine,  is  distributed  as  uniformly  as  possible  into  test-tubes 
of  equal  diameters,  each  containing  15  c.c.  of  0-1%  HC1,  the 
pepsin  solution  to  be  tested  being  then  added.  The  fibrin  stained 
with  carmine  is  dissolved  and  thus  reddens  the  liquid;  the  inten- 
sity of  the  red  colour  indicates  approximately  the  extent  of  the 
digestion.  The  digested  liquid  is  compared  with  a  number  of 
tubes  containing  carmine  solutions  of  definite  dilutions,  which 
are  so  chosen  that  the  pepsin-content  is  proportional  to  the  num- 
bers of  the  colour  scale.  This  method  has  been  modified  by 
Roaf  (Bio-Chemical  Journ.,  1908,  3,  188). 

2.  Measurement    of    the    electrical    conductivity    was    first 
employed   for   the   study   of   peptic   digestion   by   Sjoqvist 
(cf.  p.  176).     This  method  was  subsequently  largely  used  and  was 
applied  in  investigations  on  tryptic  digestion  by  Henri    and 
by  B  a  y  1  i  s  s    (Journ.  of  PhysioL,  1907,  36,  221)  and  on  the 
hydrolysis  of  dipeptides  by  erepsin  (E  u  1  e  r,  see  p.  188);   in  the 
last  experiments,  a  similar  quantity  of  alkali  solution  was  added 
to    each   solution   in   order   to   increase   the   variation   of    the 
conductivity. 

3.  Valid  objections  have  been  raised  against  the  suggestion 
made  by  S  p  r  i  g  g  s    (cf.  p.  181)  to  determine  the  progress  of 
proteolysis  by  measuring  the  viscosity  of  .the  protein  solutions, 
and  this  method  cannot  be  recommended. 

4.  A  purely  chemical  method  of  general  applicability  and 
great  accuracy  was  proposed  several  years  ago  by  Sorensen 
(Biochem.  Z.,  1908,  7,  45). 

By  this  method,  the  content  of  protein  or  its  decomposition 
products  in  a  solution  is  determined  from  the  number  of  free 
carboxyl-groups.  The  latter  can  be  estimated  by  titration  if 


APPENDIX  297 ' 

the  free  amino-groups  of  the  protein  are  first  combined.  This 
is  readily  effected  by  addition  of  excess  of  formaldehyde,  which 
unites  with  the  amino-groups,  giving  methylene-compounds. 
The  increase  of  carboxyl-groups  represents  the  extent  of  pro- 
teolysis,  which  can  hence  be  expressed  by  the  number  of  c.c. 
of  N/5-barium  hydroxide  solution  employed  in  the  titration. 
On  the  assumption  that  each  carboxyl-group  formed  during 
proteolysis  corresponds  with  one  amino-group,  the  amount  of 
proteolysis  can  also  be  stated  as  milligrams  of  nitrogen,  this 
being  obtained  by  multiplying  the  number  of  c.c.  of  the  N/5- 
barytaby  2-8. 

The  titration  is  best  carried  out  in  presence  of  thymolphthalein 
as  indicator,  the  solutions  used  being  as  follows: 

(a)  0-5  grm.  of  thymolphthalein  (G  r  ii  b  1  e  r  '  s)  dissolved 
in  1  litre  of  93%  alcohol. 

(6)  50  c.c.  of  commercial  formaldehyde  solution  are  mixed 
with  25  c.c.  of  absolute  alcohol  and  5  c.c.  of  the  thymolphthalein 
solution,  N/5-baryta  being  then  added  until  a  faint  green  or 
blue  colour  results;  this  solution  should  be  prepared  fresh  for 
each  series  of  experiments. 

As  a  control  solution,  20  c.c.  of  boiled  water  are  used.  To 
this  are  added  15  c.c.  of  the  formaldehyde  solution  (b)  and  about 
5  c.c.  of  the  baryta  solution,  the  liquid  being  then  titrated  back 
with  N/5-HC1  until  it  assumes  a  faint  blue  opalescence.  An 
addition  is  then  made  of  two  drops  of  baryta,  which  should  change 
the  colour  to  a  distinct  blue,  and  finally  of  two  further  drops, 
which  should  produce  a  vivid  blue  colour. 

It  is  this  last  colour  which  is  obtained  in  titrating  the  protein 
solution,  20  c.c.  of  which  is  mixed  with  15  c.c.  of  the  formalde- 
hyde solution  (6)  and  a  slight  excess  of  baryta  solution;  it  is 
then  titrated  back  with  HC1  until  the  colour  is  fainter  than  that 
of  the  control  solution,  the  baryta  solution  being  finally  added 
in  drops  until  the  deep  blue  of  the  control  is  obtained. 

For  the  description  of  the  titration  with  phenolphthalein, 
the  original  paper  must  be  consulted. 

The  methods  of  V  o  1  h  a  r  d  (Munch.  Med.  Wochens.,  1903, 
No.  49)  and  L  6  h  1  e  i  n  (Hofm.  Beitr.,  1905,  7,  120)  are  based 
on  methods  given  by  T  h  o  m  a  s  and  Weber,  and  by  M  e  u  - 
nier  (1901).  In  both,  casein  is  employed;  Thomas  and 
Weber  dissolve  100  grms.  of  casein  in  1900  c.c.  of  water  with 


298  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

the  aid  of  3-2  grms.  of  sodium  hydroxide  (  =  80  c.c.  normal 
NaOH)  or  5-04  grms.  of  hydrochloric  acid  (  =  138  c.c.  normal 
HC1).  The  alkaline  and  acid  solutions  serve  for  the  estimation 
of  trypsin  and  pepsin  respectively.  After  the  digestion,  the 
liquid  is  acidified,  if  necessary,  with  sulphuric  acid  and  salted 
out  with  20%  sodium  sulphate  solution.  After  filtration,  the 
precipitate  is  washed  on  the  filter  with  hot  water  until  the  last 
trace  of  sulphuric  acid  is  removed;  the  filter  and  precipitate  are 
dried  and  weighed  and  the  weight  of  undigested  protein  compared 
with  that  obtained  in  a  blank  experiment  without  pepsin  or 
trypsin.  The  amount  of  protein  dissolved  gives  a  measure  of 
the  digestive  power  of  the  gastric  juice  examined. 

In  Meunier's  method,  the  gastric  juice  (14  c.c.)  is 
mixed  with  pure  hydrochloric  acid  (0-4  c.c.)  and  casein  (1  grm.). 
After  the  casein  has  settled,  2  c.c.  of  the  clear  liquid  are  removed 
and  the  content  of  free  hydrochloric  acid  estimated.  The 
remainder  of  the  liquid  with  the  undissolved  casein  is  kept  for 
24  hours  in  a  water-bath  at  40°,  the  hydrochloric  acid  being  again 
estimated  in  2  c.c.  of  the  filtrate.  Since  hydrochloric  acid  com- 
bines with  protein  during  peptic  digestion,  the  diminution  in  the 
amount  of  the  free  acid  expresses  the  extent  of  the  action. 

V  o  1  h  a  r  d  employs  a  modification  of  the  gravimetric 
method  given  by  Thomas  and  Weber.  As  we  have  seen, 
the  latter  method  is  based  on  the  observation  that  pure,  unal- 
tered casein,  dissolved  in  the  hydrochloric  acid  of  the  digest,  is 
completely  precipitated  by  sodium  sulphate.  Hence,  if  different 
quantities  of  the  enzyme  are  allowed  to  act  on  similar  amounts 
of  casein  solution  for  equal  intervals  of  time  at  40°,  the  precipitate 
produced  by  addition  of  sodium  sulphate  will  be  the  smaller, 
the  less  the  proportion  of  casein  remaining  undigested,  i.e.,  the 
larger  the  proportion  peptonised  by  the  enzyme;  the  larger  the 
residue,  the  smaller  the  quantity  of  enzyme.  Thomas  and 
Weber  collect  the  precipitate  on  a  tared,  pleated  filter-paper, 
wash  with  distilled  water  and  dry  and  weigh.  The  difference  in 
weight  between  the  residues  from  experiments  in  which  pepsin 
has,  and  has  not  been  employed,  serves  as  a  measure  of  the  peptic 
action. 

V  o  1  h  a  r  d  avoids  the  inconvenience  of  this  weighing  by 
titration  of  the  filtrate.  He  proceeds  on  the  assumption  that 
peptonisation  of  the  casein  solution  is  accompanied  by  increase 


APPENDIX  299 

of  the  acidity  of  the  filtrate,  the  peptone  hydrochlorides  being 
non-precipitable  by  sodium  sulphate  and  hence  passing  through 
the  filter.  His  experiments  showed  that,  when  equal  quantities 
of  the  same  acid  casein  solution  without  pepsin  were  used,  the 
acidity  of  the  filtrate  was  always  constant  and  much  smaller 
than  corresponded  with  the  true  acidity  of  the  original  solution. 
This  depends  on  the  fact  that,  under  similar  experimental  con- 
ditions, the  casein  precipitate  always  contains  the  same  amount 
of  hydrochloric  acid,  only  the  free  acid  passing  into  the  filtrate. 
It  is  therefore  justifiable  to  refer  the  excess  of  acidity  over  this 
constant  value  to  the  peptone  hydrochlorides  in  the  filtrate, 
and  hence  to  regard  the  increase  of  acidity  as  a  measure  of  the 
extent  of  digestion. 

The  undigested  residues  are  in  inverse  proportion  to  the 
acidities  of  the  filtrates. 

As  example  may  be  given  the  following  results  of  V  o  1  h  a  r  d 
taken  from  L  6  h  1  e  i  n  '  s  paper  (1  o  c  .  c  i  t .)  : 

One  hundred  c.c.  of  casein  solution,  previously  heated  with  150  c.c. 
of  water,  were  digested  with  O'l,  0-4,  or  0-9  c.c.  of  gastric  juice  (acidity 
59  :  87)  for  one  hour.  Each  solution  was  then  made  up  to  300  c.c. 
in  a  graduated  cylinder  and  precipitated  with  100  c.c.  of  20%  sodium 
sulphate  solution.  Titration  of  200  c.c.  of  the  filtrate  from  the  solution 
which  contained  no  gastric  juice  in  presence  of  phenolphthalein,  gave 
the  acidity  as  19-15. 

Two  hundred  c.c.  from  the  other  experiments  gave 

Increase  of 
acidity. 

1.  0-lc.c.  gastric  juice,  22-25-19- 15 -acid  of  the  juice  (0-043)=3'06 

2.  0-4c.c.  ,25-5-19-15-  (0-17)    =6-18 

3.  0-9c.c.  ,28-5^19-15-      "  (0-387)=8-96 

The  casein  precipitates  were  collected  on  weighed  filters,  washed, 
completely  dried,  washed  again  and  finally  dried  until  constant  in 
weight. 

Weight  of  precipitate  from  original  solution,  A  =4- 104  grms. 

1  3-607    " 

2  3-053    " 

3  2-585    " 

The  amount  digested  is  hence,  bv  1  (0-1  c.c.)  A—I  =0-497  grm. 

'   2  (0-4  c.c.)  A -2  =  1-051  grms. 
"   3  (0-9  c.c.)  A  -3  =  1-519    '' 


300  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

The  proportionality  between  the  degree  of  acidity  of  the  nitrate 
and  the  digested  amount  determined  by  weighing,  is  shown  by  the 
quotients: 


Of  the  investigations  in  which  this  method  has  been  widely 
used,  that  ofS.Kuttner  (H.,  1907,  52,  63)  may  be  mentioned. 

In  the  practical  application  of  Volhard's  method,  the 
formulation  of  the  results  proposed  by  V  o  1  h  a  r  d  himself  is 
to  be  recommended.  The  pepsin-unit  is  taken  to  be  that  quan- 
tity of  enzyme  which  renders  the  nitrate  from  the  whole  of  the 
casein  used  more  acid  by  1  c.c.  of  decinormal  acid. 

The  digestion  which  would  be  produced  by  1  c.c.  of  gastric  juice  in 
1  hour  is  found  from  the  experimental  numbers  by  dividing  the  increase 
of  acidity  by  the  product  of  the  time,  t,  and  number  of  c.c.  of  juice 
employed,  /.  This  number  is  to  be  multiplied  by  2  or  by  4,  according 
as  200  or  100  c.c.  of  the  nitrate  are  titrated.  The  values  thus  obtained 
for  the  increase  of  acidity  follow  S  c  h  ii  t  z  '  s  rule,  the  pepsin-unit 
being  given  by  the  formula, 

_v*_ 

'"f-f 

Example:  Suppose  the  acidity  of  200  c.c.  of  the  original  solution, 
after  precipitation  and  nitration,  is  18*0,  i.e.,  36-0  per  400  c.c.,  and 
that  of  the  juice,  20  c.c.  per  100  c.c.  of  juice.  Then,  if  in  the  digestion 
of  100  c.c.  of  casein  solution  made  up  to  300  c.c.  with  3  c.c.  of  gastric 
juice  for  3  hours,  200  c.c.  of  the  nitrate  obtained  after  adding  100  c.c. 
of  sodium  sulphate  solution  show  an  acidity  of 

32 -7(  =65-4  per  400  c.c.), 
the  calculation  is  as  follows: 

less  36-0,  for  the  original  solution, 
and  less    0-6,  for  the  gastric  juice 


v=28-8 

OQ  •  C 


o  Xo 


=  3  •  2,     and    x  =  10  •  24  pepsin-units. 


Two  simple  methods,  apparently  well  suited  to  the  clinical 
estimation  of  pepsin,  are  due  to  J  a  c  o  b  y  and  F  u  1  d  . 

According  to  Jacoby's    method   (Biochem.  Z.,   1906,  1, 


APPENDIX  301 

58),  0-5  grm.  of  ricin  is  dissolved  in  5%  sodium  chloride  solution 
and  filtered.  An  opalescent  solution  is  obtained  which  becomes 
turbid  on  addition  of  decinormal  hydrochloric  acid.  Equal 
volumes  of  this  solution  are  mixed  with  diminishing  quantities 
of  differently  diluted  gastric  juice  and  then  made  up  with  dis- 
tilled water  or  boiled  gastric  juice  to  a  constant  volume.  After 
3  hours  in  a  thermostat,  the  liquids  are  examined  to  ascertain 
the  smallest  quantity  of  gastric  juice  able  to  clear  the  solution, 
i.e.,  to  digest  the  protein  present  completely. 

If,  after  dilution  of  the  juice  a  hundredfold,  1  c.c.  is  just 
sufficient  for  this  purpose,  the  number  of  pepsin-units  in  the 
original  gastric  juice  is  taken  as  100  (normal  gastric  juice  con- 
tains 100-200  pepsin -units). 

F  u  1  d  '  s  method  (F  u  1  d  and  L  e  v  i  s  o  n  ,  Biochem.  Z., 
1907,  6,  473;  see  also  Zeitschr.  klin.  Med.,  1907,  64,  376)  is  as 
follows:  A  clear  boiled  solution  (1  :  1000)  of  crystalline  edestin 
in  N/300-hydrochloric  acid  is  prepared,  the  edestin  being  thus 
converted  into  the  so-called  edestan. 

The  gastric  juice  to  be  examined  is  now  diluted  in  the  pro- 
portion 1  :  20  and  a  series  of  dry  test-tubes  charged  with  diminish- 
ing amounts  of  this  diluted  juice  by  means  of  a  1  c.c.  pipette 
reading  to  0-01  c.c.  These  tubes  should  have  a  diameter  of 
not  more  than  about  1  c.m.,  so  that  mixing  may  be  avoided  on 
subsequent  addition  of  ammonia. 

The  selected  amount,  say  2  c.c.,  of  edestin  solution  is  then 
rapidly  added  to  each  tube  and  after  a  lapse  of  30  minutes  ammonia 
solution  is  poured  carefully  into  each  tube,  starting  with  the  one 
containing  most  pepsin.  The  tubes  are  then  observed  in  incident 
light  against  a  black  background,  the  one  containing  the  smallest 
amount  of  pepsin  and  showing  no  ring  being  noted. 

The  number  of  c.c.  of  pepsin  solution  or  gastric  juice  con- 
tained in  this  tube  is  divided  by  the  product  of  its  dilution  and 
the  number  of  c.c.  of  edestin  solution  digested.  If,  therefore, 
0-25  c.c.  of  the  1  :  20  concentration  of  the  gastric  juice  is  suf- 
ficient to  prevent  the  formation  of  the  ring  in  2  c.c.  of  edestin 
solution,  the  number  required  is  0-25:20X2  =  1:160.  The 
gastric  juice  is  then  termed  a  1  :  160  pepsin  or  is  said  to  contain 
160  pepsin-units. 

The  methods  of  J  a  c  o  b  y  and  F  u  1  d  have  been  repeatedly 
tested  and  found  to  be  of  general  utility. 


302  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

Witte  (Berl.  klin.  Wochens.j  1907,  44,  1338)  suggests  in 
Jacoby's  method  a  slight  but  not  unimportant  modification : 
before  use,  the  gastric  juice  is  exactly  neutralised;  the  results 
are  thus  rendered  more  exact. 

Also  Reicher  (Wien.  klin.  Wochens.,  1907,  20,  1508) 
gives  these  methods  the  preference  over  all  the  older  quantitative 
methods.  He,  too,  found  the  influence  of  acidity  to  be  very 
considerable  and  agreed  with  W  i  1 1  e  '  s  proposal  to  neutralise 
the  gastric  juice.  S  o  1  m  s  (Zeitschr.  klin.  Med.,  1907,  64,  159) 
likewise  obtained  favourable  results  with  Jacoby's  method. 

E  i  n  h  o  r  n  (Berl.  klin.  Wochens.,  1908,  45,  1567)  suggests 
the  simplification  of  Jacoby's  method  by  the  use  of  an  appa- 
ratus of  special  construction.  This  apparatus,  which  is  a  glass 
vacuum-vessel,  contains  water  at  50-60°  and  a  stand  holding 
graduated  test-tubes  charged  with  the  digestion  mixture.  The 
time  of  a  test  may  thus  be  shortened  to  30  minutes. 

Of  the  researches  in  which  F  u  1  d  and  L  e  v  i  s  o  n  '  s  method 
has  been  successfully  employed,  those  of  Wo  1  f  f  and  von 
Tomaszewski  (Berl.  klin.  Wochens.,  1908),  45,  1051)  deserve 
special  mention. 


In  M  e  1 1 '  s  method,  hens'-egg  albumin  is  drawn  up  into 
a  glass  tube  1-2  m.m.  in  diameter  and  coagulated  in  the  tube  at 
95°,  lengths  of  about  2  c.m.  of  the  tube,  cut  sharply  off,  being 
then  immersed  in  the  peptic  liquid.  The  length  of  the  digested 
cylinder  of  the  albumin  is  measured  after  10  hours;  it  should 
not  exceed  about  6  m.m.  The  amount  of  pepsin  is  proportional 
to  the  square  of  this  length. 

According  toNierenstein  and  S  c  h  i  f  f  (Archiv.  f.  Verd. 
Krank.,  1902,  8,  559),  the  gastric  juices  to  be  compared  should 
be  brought  to  equal  degrees  of  acidity. 

These  methods,  which  are  open  to  the  objections  indicated 
on  p.  292,  resemble  that  of  F  e  r  m  i  founded  on  the  solution  of 
layers  of  solidified  gelatine. 

H  a  1 1  o  r  i  (Arch,  internat.  de  Pharm.  et  de  Therap.,  1908, 
18,  255),  points  out  that,  in  general,  gelatine  is  digested  much 
more  rapidly  than  coagulated  albumin  and  that  two  different 
enzymes  may  be  in  question.  Such  a  statement  is,  however, 
hypothetical  and  the  criticism  of  Fermi's  method  based  thereon 
insufficiently  supported. 


APPENDIX  303 

Estimation  of  Trypsin    and    Erepsin 

Most  of  the  methods  given  above  for  the  estimation  of  pepsin 
may  be  used,  with  suitable  modification,  to  follow  tryptic  diges- 
tion. This  is  the  case  with  the  optical  methods,  the  conductivity 
method  (cf.  Henri,  and  B  a  y  1  i  s  s  ,  p.  186),  etc. 

The  application  of  V  o  1  h  a  r  d'  s  method  (cf .  p.  297)  to 
the  estimation  of  trypsin  is  described  by  L  6  h  1  e  i  n  (1  o  c  . 
c  i  t .)  ;  the  only  difference  is  that  the  hydrochloric  acid  is  added 
to  the  casein  after  the  digestion,  whilst  in  the  investigation 
of  pepsin  it  is  added  before  digestion. 

A  similar  method  was  employed  by  R.  Goldschmidt 
(Deut.  med.  Wochens.,  1909,  35,  522). 

J  a  c  o  b  y  '  s  ricin  method  also  serves  for  the  estimation  of 
trypsin  (Biochem.  Z.,  1908,  10,  229).  Two  c.c.  of  a  solution  of 
1  grm.  of  Merck's  ricin  in  100  c.c.  of  1-5%  sodium  chloride 
solution  are  placed  in  each  of  a  series  of  tubes  together  with 

0,0-1,  0-2,0-3,  0-5,  0-7,  1-0  c.c. 

of  a  1%  solution  of  Griibler's  trypsin.  Water  is  added  to 
bring  the  volume  to  3  c.c.  in  each  tube,  to  which  0-5  c.c.  of  1% 
soda  solution  is  then  added.  The  tube  without  trypsin  remains 
persistently  turbid  whilst  the  others  gradually  clear,  that  with 
0-1  c.c.  of  trypsin  becoming  quite  bright  after  6  hours  in  an 
incubator. 

C  h  y  m  o  s  i  n  .  The  activity  of  a  solution  of  this  enzyme 
is  estimated  by  determining  in  what  dilution  it  just  coagulates 
a  certain  quantity  of  milk  in  30  minutes  at40°(K.Glaessner, 
Hofm.  Beitr.,  1901,  1,  1,  24;  Hammarsten,  H.,  1896,  22, 
103). 

Since  the  milk  used  for  the  estimation  of  rennet  varies  very 
considerably  as  regards  its  chymosin-content,  Blum  and 
Fuld  (Berl.  klin.  Wochens.,  1905,  42,  107;  Biochem.  Z.,  1907, 
4,  62)  propose  to  replace  the  milk  by  a  preparation  of  milk- 
powder,  which  is  prepared  commercially  and  is  of  constant 
composition.  Three  grms.  of  the  milk-powder  are  mixed  with 
9  times  the  weight  of  water  in  the  following  manner :  the  weighed 
(or  measured,  after  pressing  down  and  smoothing  in  a  measure) 
quantity  of  the  powder  is  added  in  small  portions  to,  and  stirred 


304  GENERAL  CHEMISTRY  OF  THE  ENZYMES 

with,  sufficient  distilled  water  to  form  a  semi-solid  paste,  to 
which  the  remainder  of  the  water  is  then  added.  On  stirring, 
almost  the  whole  of  the  powder  goes  into  solution  without  heating, 
and  this  solution,  which  can  be  prepared  in  a  couple  of  minutes, 
can  be  used  immediately  if  the  sediment  is  rejected.  It  will, 
on  the  other  hand,  keep  for  3  days  in  an  ice-chest.  An  addition 
of  calcium  salt  which  was  recommended  by  these  authors  in  their 
first  communication,  is  unnecessary. 

Twenty  tubes  are  now  charged  with  the  following  amounts: 

(1)  of  the  undiluted  gastric  juice, 

0-10,  0-15,  0-21,  0-32,  0-46,  0-68,  1-0  c.c.; 

(2)  of  the  dilution  1  :  10, 

0-10,  0-15,  0-21,  0-32,  0-46,  0-68  c.c.; 

(3)  of  the  dilution  1  :  100, 

0-10,  0-15,  0-21,  0-32,  0-46,  0-68  c.c. 

The  last  tube,  contained  1-5  c.c.  of  the  boiled  gastric  juice, 
serves  as  a  control. 

The  solutions  are  then  made  up  to  10  c.c.  with  the  milk  solu- 
tion, so  that  they  contain  the  gastric  juice  in  dilutions  varying 
from  1  :  10  to  1  :  10,000  (a  preliminary  test  being  thus  unneces- 
sary); the  tubes  are  then  placed  in  a  large  water-bath  at  17-5°. 

At  the  end  of  2  hours,  a  drop  of  20%  calcium  chloride 
solution  is  added  to  each  of  the  tubes,  which  are  then  transferred 
to  a  water-bath  at  40°  for  5  minutes.  The  ratio,  gastric  juice : 
milk  solution  in  the  clotted  liquid  containing  the  least  amount 
of  the  juice  gives  directly  the  rennetic  value  of  the  gastric  juice 
and  also  its  enzyme-content  in  general.  A  more  accurate  es- 
timation can  afterwards  be  made,  either  immediately  or,  if  an  ice- 
chest  is  available,  on  the  following  day. 

Z  y  m  a  s  e  .  For  technical  purposes  and,  indeed,  whenever 
great  accuracy  is  not  desired,  the  fermenting  power  of  pressed 
yeast-juice  or  permanent  yeast  is  determined  by  placing  in  a 
small  Erlenmeyer  flask  (100  c.c.)  furnished  with  a  M  e  i  s  s  1 
valve,  20  c.c.  of  the  pressed  juice,  8  grms.  of  cane-sugar  (or  2  grms. 
of  permanent  yeast,  10  grms.  of  water  and  4  grms.  of  cane-sugar), 
and  a  little  toluene,  the  loss  in  weight  being  determined  after  1, 
2,  3  or  4  days  at  22°.  The  evolution  of  carbon  dioxide  amounts 
to  about  1-2  grms.  (E.  and  H.  Buchner  and  M.  H  a  h  n  , 
Die  Zymasegarung,  1903,  p.  80). 


APPENDIX  305 

For  more  accurate  estimations,  the  carbon  dioxide  is  expelled 
from  the  solution  by  a  gentle  stream  of  air,  or  the  evolution  of 
gas  is  allowed  to  take  place  under  diminished  pressure,  the 
amount  of  carbon  dioxide  liberated  being  then  determined 
volumetrically. 

An  excellent  volumetric  method  has  been  devised  by 
S  1  a  t  o  r  (Journ.  Chem.  Soc.,  1906,  89,  128). 

Oxydase  and  peroxydase.  The  numerous  reac- 
tions which  have  been  employed  for  the  detection  and  estimation 
of  the  oxydases  have  been  referred  to  on  p.  59.  Since,  as  was 
previously  mentioned,  no  general  method  for  the  quantitative 
determination  of  the  oxydases  exists,  the  methods  given  for  the 
study  of  special  oxydases  will  not  be  described  here.  Reference 
may  be  made  to  the  brief  outlines  given  on  pp.  220-223. 

C  a  t  a  1  a  s  e.  Since  the  determination  of  the  catalase- 
content  of  the  blood  and  other  liquids  of  the  body  is  now  one  of 
the  more  common  tests  of  physiological  chemistry,  the  most 
important  methods  may  be  shortly  mentioned. 

With  aqueous  solutions  of  purified  catalases,  the  undecom- 
posed  hydrogen  peroxide  is  usually  determined  by  titration  with 
potassium  permanganate.  The  most  suitable  concentrations 
of  the  peroxide  are  N/20— N/50;  the  solutions  are  acidified 
with  sulphuric  acid  and  titrated  with  centinormal  permanganate. 
In  many  cases  this  method  is,  as  B  r  e  d  i  g  found  in  his  researches 
on  colloidal  metals,  preferable  to  measurement  of  the  volume 
of  oxygen  evolved. 

Where  the  fluid  of  an  organ  is  investigated  directly,  volume- 
and  pressure-methods  may  possess  decided  advantages.  A 
volume-method  was  employed  by  L.  von  Liebermann 
(Pflug.  Arch.,  1904,  104,  176)  and  more  recently  also  by  S  a  n - 
tesson,  while  W.  L  6  b  (Biochem.  Z.,  1908,  13,  339)  has 
described  an  arrangement,  which  apparently  allows  of  rapid  and 
accurate  measurement  of  catalase-content.  In  the  same  com- 
munication, Lob  describes  a  pressure-method,  which  also  serves 
well  in  certain  cases. 


306 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


TABLES    FOR    THE    ESTIMATION    OF    SUGARS    BY    BERTRAND'S 

METHOD 

Glucose 

4-07°X50-l  c.c. 


Fourth  crystallisation:  [a]/> 


1-960  grm.X2d.m. 


+52.' 


Sugar  in 
m  grins. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

10 

20-4 

41 

79-3 

71 

131-4 

11 

22-4 

42 

si  -i 

72 

133-1 

12 

24-3 

43 

82-9 

73 

134-7 

13 

26-3 

44 

84-7 

74 

136-3 

14 

28-3 

45 

86-4 

75 

137-9 

15 

30-2 

46 

88-2 

76 

139-6 

16 

32-2 

47 

90-0 

77 

141-2 

17 

34-2 

48 

91-8 

78 

142-8 

18 

36-2 

49 

93-6 

79 

144-5 

19 

38-1 

50 

95-4 

80 

146-1 

20 

40-1 

51 

97-1 

81 

147-7 

21 

42-0 

52 

98-9 

82 

149-3 

22 

43-9 

53 

100-6 

83 

150-9 

23 

45-8 

54 

102-3 

84 

152-5 

24 

47-7 

55 

104-1 

85 

154-0 

25 

49-6 

56 

105-8 

86 

155-6 

26 

51-5 

57 

107-6 

87 

157-2 

27 

53-4 

58 

109-3 

88 

158-8 

28 

55-3 

59 

111-1 

89 

160-4 

29 

57-2 

60 

112-8 

90 

162-0 

30 

59-1 

61 

114-5 

91 

163-6 

31 

60-9 

62 

116-2 

92 

165-2 

32 

62-8 

63 

117-9 

93 

166-7 

33 

64-6 

64 

119-6 

94 

168-3 

34 

66-5 

65 

121-3 

95 

169-9 

35 

68-3 

66 

123-0 

96 

171-5 

36 

70-1 

67 

124-7 

97 

173-1 

37 

72-0 

68 

126-4 

98 

174-6 

38 

73-8 

69 

128-1 

99 

176-2 

39 

75-7 

70 

129-8 

100 

177-8' 

40 

77-5 

APPENDIX 


307 


Invert-sugar 

A  0-5%  solution  was  prepared  by  hydrolysing  4-750  grms.  of  cane-sugar 
•with  50  c.c.  of  2%  hydrochloric  acid.  The  solution  was  heated  for  10-15 
minutes,  cooled,  neutralised,  and  diluted  to  a  litre. 


Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

10 

20-6 

41 

79-5 

71 

130-8 

11 

22-6 

42 

81-2 

72 

132-4 

12 

24-6 

43 

83-0 

73 

134-0 

13 

26-5 

44 

84-8 

74 

135-6 

14 

28-5 

45 

86-5 

75 

137-2 

15 

30-5 

46 

88-3 

76 

138-9 

16 

32-5 

47 

90-1 

77 

140-5 

17 

34-5 

48 

91-9 

78 

142-1 

18 

36-4 

49 

93-6 

79 

143-7 

19 

38-4 

50 

95-4 

80 

145-3 

20 

40-4 

51 

97-1 

81 

146-9 

21 

42-3 

52 

98-8 

82 

148-5 

22 

44-2 

53 

100-6 

83 

150-0 

23 

46-1 

54 

102-3 

84 

151-6 

24 

48-0 

55 

104-0 

85 

153-2 

25 

49-8 

56 

105-7 

86 

154-8 

26 

51-7 

57 

107-4 

87 

156-4 

27 

53-6 

58 

109-2 

88 

157-9 

28 

55-5 

59 

110-9 

89 

159-5 

29 

.57-4 

60 

112-6 

90 

161-1 

30 

59-3 

61 

114-3 

91 

162-6 

31 

81-1 

62 

115-9 

92 

164-2 

32 

63-0 

63 

117-6 

93 

165-7 

33 

64-8 

64 

119-2 

94 

167-3 

34 

66-7 

65 

120-9 

95 

168-8 

35 

68-5 

66 

122-6 

96 

170-3 

36 

70-3 

67 

124-2 

97 

171-9 

37 

72-2 

68 

125-9 

98 

173-4 

38 

74-0 

69 

127-5 

99 

175-0 

39 

75-9 

70 

129-2 

100 

176-5 

40 

77-7 

308 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Galactose 

16-03°X25c.c. 
Fifth  crystallisation:  [a]z>  =  — 

2  •  5  grms.  X  2  d.m. 


Sugar  in 
mgrms. 

Cu  in 

mgrms. 

Sugar  in 
mgrms. 

Cu  in 

mgrms. 

Sugar  in 
mgrms. 

Cu  in 

mgrms. 

10 

19-3 

41 

75-6 

71 

126-6 

11 

21-2 

42 

77-4 

72 

128-3 

12 

23-0 

43 

79-1 

73 

130-0 

13 

24-9 

44 

80-8 

74 

131-5 

14 

26-7 

45 

82-5 

75 

133-1 

15 

28-6 

46 

84-3 

76 

134-8 

16 

30-5 

47 

86-0 

77 

136-4 

17 

32-3 

48 

87-7 

78 

138-0 

18 

34-2 

49 

89-5 

79 

139-7 

19 

36-0 

50 

91-2 

80 

141-3 

20 

37-9 

51 

92-9 

81 

142-9 

21 

39-7 

52 

94-6 

82 

144-6 

22 

41-6 

53 

96-3 

83 

146-2 

23 

43-4 

54 

98-0 

84 

147-8 

24 

.     45-2 

55 

99-7 

85 

149-4 

25 

47-0 

56 

101-5 

86 

151-1 

26 

48-9 

57 

103-2 

87 

152-7 

27 

50-7 

58 

104-9 

88 

154-3 

28 

52-5 

59 

106-6 

89 

156-0 

29 

54-4 

60 

108-3 

90 

157-6 

30 

56-2 

61 

110-0 

91 

159-2 

31 

58-0 

62 

111-6 

92 

160-8 

32 

59-7 

63 

113-3 

93 

162-4 

33 

61-5 

64 

115-0 

94 

164-0 

34 

63-3 

65 

116-6 

95 

165-6 

35 

65-0 

66 

118-3 

96 

167-2 

36 

66-8     ' 

67 

120-0 

97 

168-8 

37 

68-6 

68 

121-7 

98 

170-4 

38 

70-4 

69 

123-3 

99 

172-0 

39 

72-1 

70 

125-0 

100 

173-6 

40 

73-9 

APPENDIX 


309 


Maltose 


Third  crystallisation:  [<Z]D: 


26-10°X  25c.c. 
2-5  grms.  X  2  d.m. 


The  above  rotation  refers  to  the  hydrate, 
lowing  tables  to  the  anhydrous  sugar. 


but  the  fol- 


Sugar in 
mgrms. 

Cu  in 

mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

10 

11-2 

40 

44-1 

70 

76-5 

11 

12-3 

41 

45-2 

71 

77-6 

12 

13-4 

42 

46-3 

72 

78-6 

13 

14-5 

43 

47-4 

73 

79-7 

14 

15-6 

44 

48-5 

74 

80-8 

15 

16-7 

45 

49-5 

75 

81-8 

16 

17-8 

46 

50-6 

76 

82-9 

17 

18-9 

47 

51-7 

77 

84-0 

18 

20-0 

48 

52-8 

78 

85-1 

19 

21-1 

49 

53-9 

79 

86-1 

20 

22-2 

50 

55-0 

80 

87-2 

21 

23-3 

51 

56-1 

81 

88-3 

22 

24-4 

52 

57-1 

82 

89-4 

23 

25-5 

53 

58-2 

83 

90-4 

24 

26-6 

54 

59-3 

84 

91-5 

25 

27-7 

55 

60-3 

85 

92-6 

26 

28-9 

56 

61-4 

86 

93-7 

27 

30-0 

57 

62-5 

87 

94-8 

28 

31-1 

58 

63-5 

88 

95-8 

29 

32-2 

59 

64-6 

89 

96-9 

30 

33-3 

60 

65-7 

90 

98-0 

31 

34-4 

61 

66-8 

91 

99-0 

32 

35-5 

62 

67-9 

92 

100-1 

33 

36-5 

63 

68-9 

93 

101-1 

34 

37-6 

64 

70-0 

94 

102-2 

35 

38-7 

65 

71-1 

95 

103-2 

36 

39-8 

66 

72-2 

96 

104-2 

37 

40-9 

67 

73-3 

97 

105-3 

38 

41-9 

68 

74-3 

98 

106-3 

39 

43-0 

69 

75-4 

99 

107-4 

100 

108-4 

310 


GENERAL  CHEMISTRY  OF  THE  ENZYMES 


Fifth  crystallisation:  [<Z]D 


Lactose 
13 


2-5  grms.XS  d.m' 


The  above  rotation  refers  to  the  hydrate,  Ci2H22Oii+H2O,  but   the  fol- 
lowing tables  to  the  anhydrous  sugar. 


Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cuin 
mgrms. 

10 

14-4 

41 

56-7 

71 

95-4 

11 

15-8 

42 

58-0 

72 

96-6 

12 

17-2 

43 

59-3 

73 

97-9 

13 

18-6 

44 

60-6 

74 

99-1 

14 

20-0 

45 

61-9 

75 

100-4 

15 

21-4 

46 

63-3 

76 

101-7 

16 

22-8 

,47 

64-6 

77 

102-9 

17 

24-2 

48 

65-9 

78 

104-2 

18 

25-6 

49 

67-2 

79 

105-4 

19 

27-0 

50 

68-5 

80 

106-7 

20 

28-4 

51 

69-8 

81 

107-9 

21 

29-8 

52 

71-1 

82 

109-2 

22 

31-1 

53 

72-4 

83 

110-4 

23  ' 

32-5 

54 

73-7 

84 

111-7 

24 

33-9 

55 

74-9 

85 

112-9 

25 

35-2 

56 

76-2 

86 

114-1 

26 

36-6 

57 

77-5 

87 

115-4 

27 

38-0 

58 

78-8 

88 

116-6 

28 

39-4 

59 

80-1 

89 

117-9 

29 

40-7 

60 

81-4 

90 

119-1 

30 

42-1 

61 

82-7 

91 

120-3 

31 

43-4 

62 

83-9 

92 

121-6 

32 

44-8 

63 

85-2 

93 

122-8 

33 

46-1 

64 

86-5 

94 

124-0 

34 

47-4 

65 

87-7 

95 

125-2 

35 

48-7 

66 

89-0 

96 

126  5 

36 

50-1 

67 

90-3 

97 

127-7 

37 

51-4 

68 

91-6 

98 

128-9 

38 

52-7 

69 

92-8 

99 

130-2 

39 

54-1 

70 

94-1 

100 

131-4 

40 

55-4 

APPENDIX 


311 


Third  crystallisation:  [a]z> 


Mannose 

3-48°X25c.c. 
1-25  grm.X  5d.m 


=  +13-92°(£=21°), 


Sugar  in 
mgrms. 

Cu  in 
mgrms. 

Sugar  in 
mgrms. 

Cu  in 
mgrms. 

10 

20-7 

60 

113-3 

20 

40-5 

70 

130-2 

30 

59-5 

80 

146-9 

40 

78-0 

90 

163-3 

50 

95-9 

100 

179-4 

INDEX   OF  AUTHORS 


Abderhalden,  9,  33,  38,  39,  40,  41, 

43,  64,  103,  107,  116,  119,  142,  143, 

190,  192,  193,  195,  196,  228,  236, 

276,  277,  287,  293 
Abelous,  69,  265 
Aberson,  209,  210,  241 
Achalme,  269,  271 
Acree,  264 
Agulhon,  116 
Albert,  57 
Armstrong,  E.  F.,  18,  21,  22,  23,  53, 

141,  165,  166,  168,  170,  173,  174, 

261,  262 
Armstrong,   H.   E.,    11,   22,   23,   97, 

128,  150,  161,  166,  170,  174,  275 
Arrhenius,  75,  76,  131,  133,  134,  135, 

136,  147,  151,  178,  183,  200,  202, 

232,  236,  237,  267,  272 
Arthus,  11,  116 
Ascoli,  14,  15,  38,  268,  270 
Asher,  117,  120 
Aso,  59 
Auld,  22,  24,  30,  101,  140,  172,  173, 

174,  239,  262,  295 

Bach,  61,  63,  64,  65,  66,  67,  68,  69, 

108,  128,  217,  219,  220,  224,  225, 

226,  228,  246 
Baker,  14 

Bang,  36,  45,  105,  109,  201,  202,  295 
Barendrecht,  165 
Barker,  74 
Bashford,  272 
Battelli,  63,  64,  67,  70,  272 
Baur,  10 
Bayliss,  36,  37,  84,  91,  103,  104,  106, 

128,  142,  186,  241,  262,  266,  270, 

296,  303 
Beam,  123,  238 
Bechhold,  84 
Behrend,  11 

Beijerinck,  14,  19,  28,  30 
Beitzke,  266,  268 
Benjamin,  48 
Berg,  29,  95 


Bergell,  36,  249,  268,  269. 

Bergmann,  von,  271 

Berkeley,  194 

Bernard,  Claude,  14 

Bertarelli,  267,  268 

Bertrand,  21,  33,  61,  62,  64,  66,  105, 

107,  108,  221,  275,  293 
Bezzola,  270 
Bial,  15 

Bickel,  249,  250 
Biedermann,  13 
Biernacki,  237 
Bierry,  24,  28,  83,  87,  91,  100,  109, 

171,  275 
Blake,  85 
Bliss,  35 
Bloch,  41 
Blum,  269,  303 
Blunt,  246 
Bodenstein,  128,  129,  131,  152,  159, 

252,  256,  263 
Bokorny,    171 
Boldyreff,  10,  93 
Bolin,  62,  66,  79,  83,  221 
Bonfanti,  14,  15 
Bordet,  116,  272 
Borissow,  176 
Bosshard,  280 
Bourquelot,  5,  13,  18,  19,  21,  22,  23, 

24,  28,  30,  32,  59,  98 
Boysen-Jensen,  52 
Braun,  11,  268 
Breandat,  30 

Bredig,  67,  69,  122,  282,  283,  305 
Brieger,  270 
Brown,  A.,  128,  162 
Brown,  H.  T.,  13;  14,  15,  19,  128,  155, 

157 

Briicke,  35 
Brugsch,  10 
Bruno,  92 
Bruschi,  48 
BuchnerE.,  3,  5,  41,  51,  52,  53,  57, 

58,  60,  81,  83,  84,  94,  105,  116,  117, 

118,  119,  211,  288,  304 


314 


INDEX  OF  AUTHORS 


Buchner,  H.,  57,  304 

Buckmaster,  91 

Buglia,  93 

Burian,  44,  59,  119,  230 

Butkewitsch,  14,  16,  43,  100 

Caemmerer,  107,  116,  119 

Caldwell,  22,  23,  39 

Campbell,  17 

Camus,  11,  120,  240,  269 

Carlson,  15 

Cathcart,  270 

Chanoz,  12 

Charrin,  270 

Chigin,  196 

Chittenden,  39,  98,  109,  120 

Chocensky,  56 

Chodat,  48,  63,  64,  65,  66,  67,  143, 

224,  225,  240,  241 
Chodschajew,  83 
Chrzascz,  14 
Clark.  25,  291,  292 
Claus,  55 
Coehn,  85 
Coggi,  269 
Cohnheim,  J.,  16 
Cohnheim,  O.,  38,  55,  91,  92 
Cole,  97,  98,  110,  112 
Connstein,  10,  11,  149 
Courtauld,  22,  23 
Cramer,  123,  238 
Cremer,  264 
Czapek,  13 
Czyhlarz,  von,  59,  66 

Dakin,  12,  42,  279,  280 

Dam,  van,  105 

Danilewski,  92,  113,  265 

Davidsohn,  160,  161 

Dean,  18 

Delbruck,  11 

Deleano,  66,  84 

Delezenne,  91,  93,  106,  244,  270 

Detmer,  158,  291 

Devaux,  32 

Dietz,  10,  152,  256,  263 

Dietze,  102 

Doblin,  270 

Donath,  92,  93 

Donnan,  74 

Dony-Henault,  61,  62,  220 

Downes,  246 

Dox,  31 

Doyon,  11,  12 

Dreyer,  247 

Drjewezki,  von,  101 

Duchacek,  54,  57 

Duclaux,  5,  116,  117,  158 

Dunstan,  30,  295 


Durham,  108 
Duuren,  van,  61,  220 

Edelstein,  249 

Edmunds,  46 

Effront,  13,  17,  97,  109,  110,  116,  158, 
239 

Ehrenreich,  82 

Ehrlich,  44,  223 

Einhorn,  302 

Elvove,  12,  69,  146 

Embden,  55 

Emmerling,  39,  245,  261,  264 

Engel,  10,  147,  289 

Engler,  137 

Erben,  37 

Eriksson,  122,  123 

Ernest,  56 

Euler,  13,  25,  26,  27,  41,  62,  66,  67. 
68,  79,  83,  88,  100,  102,  104,  105, 
107,  112,  113,  116,  118,  121,  137, 
142,  148,  165,  188,  193,  206,  209, 
214,  216,  218,  221,  231,  232,  236, 
241,  259,  283,  296 

Faitelowitz,  67,  106,  116,  119 

Fa jans,  282 

Falloise,  10 

Falta,  250 

Farr,  119 

Faubel,  186 

Fermi,  24,  25,  83,  117,  245,  269,  271, 

Fernbach,  83,  108,  114,  264 

Finsen,  245 

Fischer,  E.,   18,   19,  21,  22,  25,    29 

30,  31,  36,  53,  79,  115,  118,  119, 

168,  261,  276,  277,  279,  283,  285, 

288 

Fischer,  W.,  24,  283 
Foa,  98 
Ford,  93,  97 
Foster,  15 
Frankel,  17 
Franzen,  52,  281 
Freederickz,  64 
Freudenreich,  118 
Freundlich,  73,  76,  88 
Freidenthal,  35,  273,  287 
Fromme,  10 

Furth,  von,  59,  66,  93,  228 
Fuhrmann,  11,  31 
Fuld,  48,  49,  119,  201,  205,  241,  272, 

300,  301,  302,  303 

Gatin,  70 
Gaunt,  60 
Gengou,  116,  272 
Gerard,  11,  23,  24 


INDEX  OF  AUTHOKS 


315 


Gerber,  48,  49,  104,  109,  111,  202, 

241,  244 

Geret,  40,  41,  119 
Gerhartz,  249 
Gessard,  272 
Gewin,  47 
Giaja,  24,  28,  275 
Gigon,  108,  142,  143 
Gizelt,  120 
Gjaldbak,  265 
Glaessner,  270,  303 
Glendinning,  128,  140,  155,  157,  264 
Gley,  19,  270 
Glimm,  238 
Glinski,  292 
Glover,  161,  166,  170 
Gockel,  120 
Godlewski,  55 
Golden,  31 

Goldschmidt,  H.,  144 
Goldschmidt,  R  ,  303 
Gonnermann,  24,  64 
Gottlieb,  70 
Grafe,  228 
Green,  Reynolds,  11,  13,  16,  18,  39, 

49,  96,  247 
Gries,  95 
Grignard,  279 
Grigoriew,  210 
Gross,  180 
Grass,  82 

Griitzner,  96,  117,  296 
Gudzent,  250 
Guggenheim,  64,  228,  236 
Guignard,  23,  29,  244 
Gulewitsch,  36 
Guthrie,  93 


Hagglund,  75 

Haehn,  54 

Hafner,  26 

Hahn,  40,  41,  57,  95,  119,  250,  269, 

304 

Hairs,  30 
Hall,  92 
Halliburton,  15 
Hamburg,  17 

Hamburger,  14,  19,  30,  91,  287 
Hammarsten,  36,  45,  46,  47,  48,  49, 

50,  83,  97,  104,  200,  205,  272,  303 
Handovsky,  85,  87 
Hanriot,  11,  128,  240,  263 
Hansen,  19,  201 
Hanssen,  247 
Harden,  3,  32,  53,  54,  56,  94,   109, 

206,  211,  213,  214,  280 
Hardy,  89 
Hart  ,'31 
Hattori,  302 


Hedin,  37,  77,  78,  81,  82,  101,  122, 

128,  187 
Heffter,  69 
Hekma,  30,  91 
Henneberg,  115 
Henri,  87,  91,  128,  129,  130,  132,  140, 

141,  156,  158,  159,  163,  165,  167, 

175,  184,  249,  252,  253,  296,  303 
Henriques,  265 
Henry,  24,  30,  262,  295 
Herissey,  13,  21,  22,  23,  24,  28,  30, 

32,  118,  168 
Herlitzka,  35 
Heron,  19 
Hertel,  245 
Herzog,  79,  137,  167,  172,  196,  210, 

240,  241,  265,  269 
Higuchi,  49 

Hildebrandt,  101,  267,  268 
Hill,  Croft,  4,  20,  83,  168,  257,  261, 

262 

Hinkins,  264 
Hober,  88,  122 
Hoff,  van't,  138,  231,  251,  253,  254, 

257,  261 

Hoffmann,  96,  117 
Hofmeister,  87,  89 
Holderer,  21,  82 
Homfray,  Miss,  75 
Hongardy,  91 
Horton,  22,  23,  174 
Hoyer,  11,  94,  97,  108,  111,  149 
Huber,  116 
Hubert,  83 
Hudson,  18,  19,  21,  26,  27,  90,  98, 

99,  100,  114,   127,   159,  160,  161, 

163,  165,  171,  237,  238,  262 
Huerre,  243 
Huiskamp,  50 
Huppert,  179,  293 

Issaew,  25,  216,  217 
Italic,  van,  215 
Iwanoff,  42,  214 
Izar,  109 

Jacobson,  105 

Jacoby,  35,  36,  83,  84,  272,  300,  301, 

302,  303 
Jalander,  152 
Jamada,  246,  248 
Jastrowitz,  136 
Jerusalem,  228 
Jochmann,  37,  269 
Jodlbauer,  246,  247,  248 
Johansson,  209 
Johnston,  146 
Jones,  42,  44 
Jones,  G.  C.,  291 
Jonescu.  244 


316 


INDEX  OF  AUTHORS 


Jorissen,  30 
Johnson,  292 
Jungk,  292 

Kalaboukoff,  9,  93 

Kalanthar,  21 

Kanitz,  10,  102,  107,  148,  149,  287 

Kantorpwicz,  269 

Karamitsas,  248 

Kasarnowski,  79 

Kastle,  12,  25,  59,  67,  69,  116,  117, 
118,  122,  128,  146,  147,  215,  224, 
225,  239,  241,  244,  263,  275,  276 

Kaufmann,  116,  118,  119 

Kayser,  108 

Kendall,  156,  264,  291,  292 

Kiesel,  43 

Kikkoji,  42,  43,  101 

Kirchoff,  15 

Kjeldahl,  117,  158,  241,  242 

Klatte,  81,  84 

Klempin,  157 

Knauthe,  13 

Kobert,  24 

Koelker,  103,  190,  192,  193,  295 

Kottlitz,  205 

Kohl,  246,  264 

Kossel,  42 

Kostytschew,  55,  56 

Kotake,  70 

Krafft,  79 

Krawkow,  16 

Krober,  118,  168,  240 

Kriiger,  25,  110 

Kudo,  102,  110 

Kiihne,  139,  142 

Kuttner,  300 

Kullberg,  25,  26,  79,  104,  121,  165, 
214,  236,  243 

Kurajeff,  265 

Kussmaul,  268 

Kutscher,  39,  42 

Laborde,  30 

Lalou,  175 

Landsteiner,  270 

Langley,  94,  98 

Lanzenberg,  108 

Laqueur,  93,  120 

Larguier  des  Bancels,  184 

Larin,  95 

Lawrow,  265 

Lea,  43 

Lebedew,  von,  57,  214 

Lesser,  66,  67,  215 

Leube,  43 

Leuchs,  15 

Levaditi,  270 

Levene,  42 

Levison,  301,  302 


Levites,  110 

Lewkowitsch,  10 

Liebermann,  von,  67,  215,  305 

Lindberger,  102 

Lindet,  63 

Lindner,  19,  240 

Ling,  14,  290 

Lintner,  16,  97,  100,  110,  118,  168, 
288,  290 

Lippmann,  von,  5 

Lobassow,  196 

Lockemann,  37,  110,  246,  249 

Loeb,  Jacques,  107,  115,  184 

Loeb,  L.,  50 

Lob,  W.,  305 

Lohlein,  186,  297,  299,  303 

Lorcher,  104 

Loevenhart,  12,  63,  67,  92,  93,  116, 
117,  118,  122,  128,  146,  147,  215, 
224,  225,  239,  241,  263,  275,  276 

Loew,  16,  67,  107,  215,  217 

Loewenherz,  275 

Loewenthal,  249 

Lohmann,  39 

London,  195,  198,  200 

Luckhardt,  15 

Lundeqvist,  137 

Luther,  137,  287 

McCollum,  31 

MacGillawry,  13 

McKenzie,  279,  280 

Maclean,  56 

Macleod,  10 

Madsen,  203,  245,  272 

Magnus,  12,  92,  93,  273 

Malfitano,  106 

Malleyre,  33,  107 

Mangin,  32 

Maquenne,  14,  155,  264 

Marchand,  108 

Marckwald,  278,  279,  283 

Martin,  205,  237 

Martinand,  62,  66 

Martini,  101 

Maxwell,  71 

Mayer,  9,  249 

Mays,  37 

Medigreceanu,  42 

Medwedew,  128,  219 

Meisenheimer,  3,  5,  51,  52,  58,  60,  94, 

211 

Meltzer,  236 
Mendel,  15 
Mett,  222 
Meunier,  297,  298 
Meyer,  Kurt,  180,  271 
Michaelis,  36,  77,  78,  82,  84,  85,  86, 

87,  160,  161,  190,  267,  287 
Millner,  73 


INDEX  OF  AUTHORS 


317 


Minami,  250 
Miquel,  240 
Moitessier,  66 
Moll,  43,  271 
Montesano,  24,  25 
Moore,  139 
Moraczewski,  von,  95 
Morawitz,  50,  91,  107 
Morel  11 

Morgenroth,  271,  272 
Moritz,  13,  140,  264 
Morris,  13,  14,  15 
Mouton,  244 
Miiller,  Ed.,  293 
Muller-Thurgau,  158,  240 
Muntz,  118 

Nagayama,  38 

Nasse,  15,  119 

Neilson,  110 

Nencki,  34,  35,  47,  92 

Neppi,  38 

Neuberg,  10,  23,  65,  249,  266,  268 

Nicloux,  11,  151,  236 

Niebel  19,  25 

Nierenstein,  302 

Norris,  53 

Novy,  35 

Niirnberg,  265 

Obermayer,  296 

Ohlsen,  214 

Okuneff,  265 

Oppenheimer,  C.,  287 

Oppenheimer,  S.,  69 

Ormerod,  97,  150,  275 

Osaka,  137 

Osborne,  17,  25,  26,  109 

Oshima,  26 

Ostwald,  Wilh.,  72,  129,  234,  287 

Ostwald,  Wo.,  72,  246,  287 

O'Sullivan,  26,  98,  158,  159,  161,  162, 

165,  237,  241,  242,  262,  288 
Overton,  264 

Pagenstecher,  10 

Paine,  21,  100,  114,  171,  237,  238 

Palladin,  55,  56 

Pantanelli,  264 

Parnas,  70 

Parrozzani,  8 

Pasteur,  276 

Patten,  114 

Pauli,  85,  87,  88,  89 

Pawlow,  34,  36,  37,  47,  91,  92,  106, 

111,  113,  157,  176,  276,  292 
Payen,  15 
Peirce,  154 
Pekelharing,  34,  35,  47,  49,  50,  86 


Pernossi,  83,  117,  245 

Persoz,  15 

Pewsner,  198 

Pfleiderer,  94,  96,  117 

Philoche,  157,  167 

Pick,  15,  296 

Pinkussohn,  107,  116,  118,  119 

Plimmer,  30 

Pohl,  59 

Pollak,  38,  102,  123,  184 

Pomeranz,  257 

Porter,  123 

Posternak,  88 

Pottevin,  10,  14,  22,  107,  263 

Pozerski,  244 

Pozzi-Escot,  68 

Preti,  110 

Price,  231 

Pringsheim,  43 

Pugliese,  15,  118,  269 

Quincke,  80 

Rachford,  92,  93 

Rapp,  105 

Raudnitz,  67 

Reichel,  94,  118,  120,  201 

Reicher,  302 

Reinhard,  63 

Reiss,  67 

Resenscheck,  84 

Rey-Pailharde,  de,  68 

Ribaut,  265 

Richter,  249 

Rinckleben,  57 

Ringer,  86 

Roaf,  296 

Roberts,  291 

Robertson,  25,  104,  266 

Roden,  272 

Rohmann,  20,  59 

Roger,  15,  109 

Rogozinski,  66 

Rona,  78,  84,  97 

Rosenberg,  108 

Rosenblatt,  64 

Rosenfeld,  63,  120,  265 

Rosenheim,  10,  92,  93 

Rosenthaler,  5,  22,  23,  45,  173,  264 

280,  281 
Rostocki,  33 
Roth,  287 
Rouge,  11,  48,  148 
Rowland,  101,  288 
Rozenband,  105 

Sachs,  101,  269 
Saiki,  15,  268 
Sailer,  119 
Salaskin,  265 


318 


INDEX  OF  AUTHORS 


Salazar,  100,  171 

Salkowski,  25,  26,  41 

Samojloff,  176 

Santesson,  117,  305 

Sarthou,  63 

Sawitsch,  47 

Saw j  alow,  265 

Schade,  52 

Schaeffer,  36,  83,  87,  110 

Schaer,  59 

Schapirow,  92 

Schardinger,  68,  69 

Schellenberg,  13 

Schiff,  302 

Schilow,  137 

Schittenhelm,  41,  43,  44 

Schlesinger,  18,  118 

Schmidt,  Alex.,  49 

Schmidt,  C.  L.  A.,  104 

Schmidt,  G.  C.,  75,  76,  78 

Schmidt-Nielsen,  Signe,  36,  47,  236, 

247 
Schmidt-Nielsen,    Sigval,    236,    247, 

248,  249 

Schmiedeberg,  12,  59 
Schneegans,  27 
Schondorff,  120 
Schorstein,  13 
Schreiner,  69 
Schrumpf,  35 

Schiitz,  E.,  132,  133,  175,  179,  295 
Schiitz,  J.,  93,  110,  179,  285 
Schiitze,  268,  269 
Schutzenberger,  28 
Schulze,  280 

Schumoff-Siemanowski,  9 
Schumoff-Siemanowski,  Mme.,  34 
Schunck,  29 
Schwarz,  123,  250 
Schwarzschild,  36,  37 
Schwiening,  101 
Scurti,  8 
Segelke,  201 
Senter,  67,  105,  106,  117,  119,  215, 

216,  217,  236,  241 
Shaklee,  236 
Shaw-Mackenzie,  93 
Shedd,  59 

Sherman,  18,  156,  264,  291,  292 
Shibata,  43 
Shiga,  43 
Shigeji,  49 
Shore,  19 

Sieber,  9,  34,  35,  47 
Sieber,  Mme.,  55 
Siedentopf,  80 
Sigmund,  11,  28,  117,  149 
Simnitzki,  270 

Sjoqvist,  95,  96,  176,  178,  296 
Slator,  51,  53,  210,  240,  243,  274,  305 


Slowtzoff,  63,  220 

Sorensen,  27,  90,  96,  98,  99,  100,  160, 

161,  165,  184,  219,  266,  296 
Solms,  302 
Sommer,  69 
Souder,  93 
Soxhlet,  201 
Spatzier,  29 
Spiro,  48,  94,  118,  119,  120,  201,  203, 

272 

Spitzer,  59,  63 
Spohr,  231 
Spriggs,  181,  296 
Stade,  10,  147 
Stangassinger,  70 
Starling,  91,  106,  270 
Steche,  216 
Stenitzer,  von,  269 
Steppuhn,  52 
Stern,  64,  70,  272 
Stevens,  63 
Stiles,  114 

Stoecklin,  de,  59,  62,  65,  66,  223 
Stoklasa,  56 
Stoll,  12 
Stone,  16 
Storch,  201 
Strada,  83 
Sullivan,  69 
Sundberg,  35 
Suzuki,  31 


Takaishi,  31 

Takamine,  16 

Takemura,  40 

Tammann,  139,  158,  171,  173,  233, 

240,  241,  242,  255,  258 
Tanret,  Ch.,  29,  262 
Tanret,  G.,  29 
Tappeiner,  von,  248 
Taylor,  47,  104,  154,  156,  157,  161, 

194,  241,  256,  257,  264,  266,  285 
Tebb,  Miss,  14,  19 
Terroine,  9,  36,  93,  167 
Terry,  110 
Teruuchi,  39 
Thierfelder,  53 
Thies,  110,  246,  249 
Thomas,  297,  298 
Tichomirow,  36 
Titoff,  75 

Tomaszewski,  von,  302 
Tompson,    98,    158,    159,   161,   162, 

165,  237,  241,  242,  262,  288 
Trebing,  270 
Trillat,  62 
Trommsdorff,  69 
Tscherniak,  65 
Tschirch,  63 


INDEX  OF  AUTHORS 


319 


Twitchel,  113 
Twort,  24 

Ugglas,  af,  27,    100,    112,    121,    165, 

241,  242,  243 
Umber,  10 

Vandevelde,  83,  109,  116,  117,  271, 

273 
Vernon,  37,  38,  46,  103,  115,  183,  223, 

237,  241,  283 
Victorow,  120 
Vines,  39,  40,  41,  100,  194 
Vinson,  25 
Visser,  242,  258 
Volhard,  10,  83,  97,  147,  288,  297, 

298,  299,  300,  303 
Vulquin,  101 

Waele,  de,  273 
Waentig,  216 
Walbum,  272 
Walker,  92 
Walther,  176,  292 
Warburg,  279 
Wartenberg,  11,  149 
WTeber,  297,  298 
Weevers,  28,  228 
Weinland,  31,  269,  271 
Weis,  39,  41,  100,  181,  246 
Welter,  264 
Wheldale,  Miss,  228 
White,  Miss,  244 


Whitney,  85 

Wichern,  110,  246,  249 

Wiechowski,  60,  288 

Wiener,  60,  288 

Wijsman,  14,  15 

Wilcock,  249 

Wilhelmy,  127 

Willstatter,  12,  108 

Windisch,  41 

Witte,  302 

Wittich,  von,  15,  16 

Wohl,  51,  238 

Wohlgemuth,  93,  94, 110, 249,291,292 

Wolff,  J.,  59,  66,  109,  223,  264 

Wolff,  W.,  302 

Wren,  279 

Wright,  16 

Wroblewski,  16,  17,  26,  96,  291 

Yoshida,  61 
Yoshimoto,  101 
Yoshimura,  31 

Young,  3,  32,  53,  54,  94,  109,  206, 
211,  213,  214 

Zahorski,  64 
Zaleski,  42,  63 
Zeller,  247,  248 
Zellner,  148,  246 
Zemplen,  21 
Zinsser,  10 
Zsigmondy,  80 
Zunz,  91,  106,  107,  271 


INDEX  OF   SUBJECTS 


Acids;  activation  by,  94 

Activators,  90  et  seq. 

Adenase,  7,  44 

Adenine,  7,^44 

Adsorption,  75  et  seq. 

Adsorption  media;  acid  or  basic,  84 
— ;  neutral,  81 

Aesculase,  28 

Alcoholic  fermentation,  51  et  seq. 

Alcoholoxydase  of  acetic  acid  bac- 
teria, 7,  60 

Aldehydases,  7,  70,  219 

Alkaloids,  119 

Amicrons,  80 

Amidases,  43 

Amygdalase,  23,  173 

Amygdalin,  22,  23,  171-175 

Amylase,  6,  13,  155 

— ;  'activation  by  acids  and  salts,  97, 
158,  289 

— ;  dynamics,  155 

— ;  occurrence,  14 

— ;  preparation,  16 

Amylopectinase,  6,  14,  155 

Anti-enzymes,  267 

Arbutase,  28 

Arginase,  42 

Arginine,  42 

Arsenic,  109,  116 

Asymmetric  syntheses,  277 

Autocatalysis,  111,  128 

Autolytic  enzymes,  101 

Bases;  activation  by,  94 

Betulase,  27 

Blood;  clotting  of,  49,  50,  205 

Borax,  29 

Boric  acid,  116 

Bromelin,  39 

"  Buffers,"  106,  115 

Butyrases,  6,  11 

Carbamases,  6,  33 
Carbonases,  56 
Caroubinase,  13 


Casein,  45,  200 

Catalase,  7,  67,  87,  116,  117,  305 

— ;  activation  by  acids  and  alkalis, 

105 

— ;  dynamics,  215 
— ;  occurrence,  67 
— ;  preparation,  67 
Catalysis,  127 
Cellase,  21 
Cellulase,  13 

Charcoal  as  adsorbent,  81 
Choral,  29,  118 
Chloroform,  118 
Chlorophyllase,  6,  12 
Chymosin,  45,  303 
— ;  activation  by  acids,  104 
— ;  dynamics,  200 
— ;  occurrence,  45 
— ;  preparation,  46 
Classification  of  enzymes,  6 
Coagulating  enzymes,  45  et  seq. 
Co-enzymes,  12,  53,  90 
Colloids,  78 
— ;  inorganic,  118 
Coupled  reactions,  137 
Creatine,  70 
Cresols,  118 
Cynarase,  48 
Cytase,  6,  13 

Desamidases,  7,  43 

Diastase,  6,  13,  97,  118,  120,  289  et 

seq. 

— ;  preparation,  16,  17 
Digestion;    dynamics  of,  195 
Dihydroxyacetone,  52 
Dynamics;  chemical,  of  reactions,  124 

'Elaterase,  29 

Electric  transference  of  enzymes,  85 
Emulsin,  6,  22,  87,  261,  266,  295 
— ;  activation  by  acids,  101 
— ;  dynamics,  171 
— ;  occurrence,  23 
— ;  preparation,  24 

321 


322 


INDEX  OF  SUBJECTS 


Endotryptase  of  yeast,  40 
Enterokinase,  91 
Enzymic  equilibria,  252 
Erepsin,  6,  38,  116,  118,  303 

—  activation  by  alkali,  102 

—  dynamics,  188 

—  occurrence,  38 

—  preparation,  38 
Erythrozyme,  29 
Esterases,  6,  9  et  seq.,  289 

—  dynamics,  146 

—  preparation,  10,  12 

Fermentation;  mechanism  of,  51 
Fermentation  enzymes,  50 
Fibrin  ferment :  see  thrombin 
Fibrinogen,  49 
Formaldehyde,  118 

Galacto-lactase,  22 
Galacto-zymase,  53 
Gastric  juice,  34,  97 
Gaultherase,  27 
Gease,  28 
Gels,  89 

Gluco-lactase,  22,  23,  170 
a-Glucosidase,  6,  19 
/3-Glucosidase,  6,  19,  21,  171 
Glucp-zymase,  53 
Glutinase,  38,  102,  184 
Glyceraldehyde,  52 
Glycerol,  118 
Glycolytic  enzymes,  55 
Guanase,  7,  44 
Guanine,  7,  44 

Hexosephosphatase,  32,  54 
Hexosephosphatese,  104,  214 
Histozyme,  12 
Hydrocyanic  acid,  119 
Hydrogen  peroxide,  67,  117 
Hydrogen  sulphide,  117 
Hypoxan thine,  44,  60 

Indigo  enzyme,  30 
Inhibiting  agents,  115 
Internal  pressure,  71 
Intestinal  juice,  101 
Inulinase,  6,  98 
—    occurrence,  18 
Invertase,  6,  24,  87,  117,  284 

activation  by  acids,  98 

destruction  by  acids  and  alkalis, 
237 

dynamics,  158 

occurrence,  25 

preparation,  25 
Isomaltose,  14 
Isomerising  enzyme,  70 


Kaolin  as  adsorbent,  84 
Kinases,  90,  91 

Laccases,  61,  62,  105 

— ;  artificial,  62 

Lactacidase,  53 

Lactase,  6,  30 

— ;  activation  by  acids,  100 

— ;  dynamics,  168 

— ;  occurrence,  30 

— ;  preparation,  31 

Lactic  acid  bacteria;  zymase  of,  7,  58 

Light;  influence  of,  on  enzymes,  245 

Linamarase,  30 

Lipases,  6,  9,  10,  116,  117,  118,  119, 

263,  264,  275,  288 
— ;  dynamics,  146 
— ;  occurrence,  10-11 
Lotase,  30 

Maltase,  6,  19-20 
— ;  dynamics,  166 
— ;  occurrence,  19 
— ;  preparation,  19 
Mandelonitrile  glucoside,  23 
Manno-zymase,  53 
Maximum  temperatures,  243 
Melibiase,  31 
Mercuric  chloride,  116 
Mercuric  cyanide,  116 
Mesothorium;    influence  of,  on  en- 
zymes, 250 
Methylglyoxal,  52 
Microns,  80 
Myrosin,  6,  29,  118 
• — ;  occurrence,  29 

Neutral  salts;  activation  by,  106 
Nitrilase,  45 
Nitrilese,  5 

Nomenclature  of  enzymes,  5 
Nuclease,  6,  41 
Nucleinases,  42 
Nucleosidases,  42 
Nucleotidases,  42 

Oenoxydase,  63 

Olease*  63 

Optimum  temperatures,  243 

Ornithine,  42 

Oxydase  of  acetic  bacteria,  60 

Oxydases,  58,  118,  219,  305 

— ;  detection,  59 

Oxynitrilase,  45 

Ozone,  117 

Pancreatic  juice,  107,  276 
Papain  or  papayotin,  39,  40,  101 
Paracasein,  45,  200 
Parachymosin,  45,  105 


INDEX  OF  SUBJECTS 


323 


Paralysors,  115 

Pectase,  6,  32 

Pectinase,  7,  32,  100 

Pepsin,  6,  33,  46,  47,  83,  116,  117,  118 

175  et  seq.,  265,  295  et  seq. 
— ;  activation  by  acids,  94  st  seq. 
— ;  dynamics,  175 
— ;  occurrence,  34 
— ;  preparation,  34 
Pepsinogen,  34 
Peptases,  40 
Permanent  yeast,  57 
Peroxydases,  7,  65,  105,  223,  305 
— ;  dynamics,  223 
— ;  preparation,  65 
Phaseolunatase,  30,  172 
Phenol,  118 
Phenolases,  7 
Philothion,  68 
Phosphates,  109 
Phosphatese,  5,  7,  104 
Phytase,  6,  31 
Plastein  formation,  265 
Poisons,  115  et  seq. 
Potassium  cyanide,  119 
Preparation  of  enzymes,  7  et  seq. 
Press  yeast  juice,  53,  54,  56 
— ;  dynamics,  206 
— ;  preparation,  56 
Prochymosin,  46 
0-Proteases,  40 
Protective  agents,  115 
Proteinases,  6,  33,  38,  295 
Proteolytic  enzymes    of    plants,    38, 

181  et  seq. 
Ptyalin,  15,  97 
— ;  preparation,  16 
Purification  of  enzymes,  7  et  seq. 

Radiation;  influence  of,  on  enzymes, 
245 

Radium;  influence  of,  on  enzymes, 
249 

Reductases  or  reducing  enzymes,  68 

Rennet:  see  chymosin 

Reversible  reactions,  137 

Revertase,  264 

Rhamnase,  6,  29 

Rontgen  rays;  influence  of,  on  en- 
zymes, 249 

Salicase,  28 
Salicylic  acid,  29,  119 
Salts,  activation  by,  106 
— ;  inhibition  by,  116 


Schinoxydase,  63 
Schiitz's  rule,  132 
Seminase,  13 
Sodium  chloride,  109 
Sodium  fluoride,  116 
Specificity  of  enzyme  actions,  274 
Spermase,  63 

Statics;  chemical,  in  enzyme  reac- 
tions, 251 

Steapsin:  see  lipase 
Stimulin,  92 
Submicrons,  80 
Sucrase:  see  invertase 
Surface  energy,  72 
Surface  tension,  72 
Syntheses  by  enzymes,  261 

Taka-diastase,  16 

Tannase,  6 

Tannin,  29 

Temperature;  influence  of,  on  en- 
zyme reactions,  231 

Temperature  coefficients  of  enzyme 
reactions,  239 

Thrombin,  7,  49,  107,  116,  205 

Thymol,  58,  118 

Toluene,  58,  118 

Transference;  electric,  85 

Trehalase,  20 

Trypsin,  6,  36,  43,  84,  86,  102,  116, 
117,  118,  119,  123,  183,  184,  186, 
194,  266,  303 

— ;  dynamics,  184 

— ;  occurrence,  37 

— ;  preparation,  37 

Tyrosinase,  64,  120,  228 

Urea,  43 

Urease,  7,  43     , 

Uric  acid,  60 

— ;  enzyme  oxidising,  41,  60 

Xanthine,  44,  60 
Yeast;  permanent,  57 

Zymase,  7,  54  et  seq.,  94,  105,  109, 

116,  118,  120,  304 
— ;  co-enzyme,  211 
— ;  dynamics,  206 
— ;  occurrence,  54 
— ;  preparation,  56,  57' 
Zymin,  58 
Zymogen,  40,  49 
— ;  activation  of,  96 


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30m-10,'61  (C3941s4)4128 


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